Fundamental characteristics of ultrasonic green formulations using Avena sativa L. extract-mediated gold nanoparticles and electroconductive nanofibers for cardiovascular nursing care.

In the pursuit of novel approaches to address chronic heart failure and enhance cardiovascular nursing care, environmentally sustainable nanomaterials have taken center stage. Recent progress in regenerative medicine has opened doors for the use of biocompatible biomaterials that provide mechanical support to damaged heart tissue and facilitate electrical signaling. This study was dedicated to developing advanced electroconductive nanofibers by incorporating eco-friendly Avena sativa L. extract-mediated gold nanoparticles (AuNPs) into polyaniline to create an intricate cardiac patch. The AuNPs were synthesized through an environmentally friendly chemical process aided by ultrasonic conditions. Comprehensive physicochemical analyses, such as UV-Vis spectroscopy, SEM, TEM, DPPH assay, and XRD, were carried out to characterize the AuNPs. These AuNPs were then blended with a polycaprolactone/gelatin polymeric solution and electrospun to fabricate cardiac patches, which underwent thorough evaluation using various techniques. The resulting cardiac patch demonstrated excellent hemocompatibility, antioxidant properties, and cytocompatibility, offering a promising therapeutic approach for myocardial infarctions and the advancement of cardiovascular nursing care.

G Protein-Coupled Receptor Kinase 4 Is a Novel Prognostic Factor in Hepatocellular Carcinoma.

Purpose: We previously reported that G protein-coupled receptor kinase (GRK) 4 halts cell cycle progression and induces cellular senescence in HEK293 cells. The present study was aimed at assessing the prognostic value of GRK4 in hepatocellular carcinoma (HCC). Methods: GRK4 expression was detected by immunohistochemistry in paired tumoral and peritumoral tissues of 325 HCC patients. One hundred and twenty-six patients from Western China were utilized as a training cohort to develop a nomogram, while 86 patients from Eastern China were used as a validation cohort. The proliferation and migration of lentiviral-GRK4 expressing HepG2 cells were determined by MTT and wound healing assays. Results: GRK4 was differentially expressed in HCC tissues. Tumoral GRK4 intensity, tumor type, and T stage were independent prognostic factors and used to form a nomogram for predicting overall survival (OS), which obtained a good concordance index of 0.82 and 0.77 in training and validation cohort, respectively. The positive and negative prediction values with nomogram were, respectively, 83% and 75% in training cohort and 100% and 52% in validation cohort. Patients with nomogram scores > 32 and 78 showed high risk for OS. Proliferation and motility capabilities were significantly restrained in GRK4-overexpressing HCC cells. Discussion. Low GRK4 expression in HCC tumor tissues indicates poor clinical outcomes. A prognostic nomogram including tumoral GRK4 expression would improve the predictive accuracy of OS in HCC patients. We also demonstrated that GRK4 overexpression inhibits proliferation and migration of HCC cells. The molecular mechanism underlying is worth further study.

Combined polymorphisms in genes encoding the inflammasome components NLRP3 and CARD8 confer risk of ischemic stroke in men.

INTRODUCTION: Previous studies have reported the involvement of nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome in the inflammatory activation and pathophysiology of Ischemic Stroke (IS). Variations in genes encoding the constituent proteins of NLRP3 inflammasome can alter the risk of IS. OBJECTIVE: We investigated the role of the NLRP3 inflammasome in the pathogenesis of IS by establishing associations between combined polymorphisms of caspase recruitment domain-containing protein 8 (CARD8) rs2043211 and NLRP3 rs10754558 and the susceptibility to IS in a Chinese population. METHODS: Single nucleotide polymorphisms (SNPs) in CARD8 rs2043211 and NLRP3 rs10754558 were analyzed using TaqMan SNP genotyping assays in patients with IS (n=234) and healthy controls (n=115). Logistic regression analysis was carried out to evaluate potential interactions between CARD8 and NLRP3. RESULTS: Compared with healthy controls, there were no significant differences in the minor allele frequency (MAF) and the genotype frequency of NLRP3 rs10754558 or CARD8 rs2043211 in patients with IS(P>0.05). After stratification by gender, there was an increased risk for IS in men carrying heterozygous CARD8 rs2043211 when a co-dominant genetic model was applied (P=0.021, OR=3.83[1.22-12.03]). Logistic regression analysis indicated that men carrying both CARD8 rs2043211 AT and NLRP3 rs10754558 CG had a significantly higher risk of IS (P=0.046, OR=7.116[1.033-49.044]). CONCLUSIONS: Nucleotide variations in the genes encoding NLRP3 inflammasome proteins may be important to IS, and men carrying CARD8 rs2043211 and NLRP3 rs10754558, both heterozygous, confer a higher risk of IS.

Spatiotemporal assessment of marine environmental monitoring programme based on DIN concentration in the Yangtze River estuary and its adjacent sea.

The marine environment is rigorously protected in the Yangtze River Estuary (YRE) and its adjacent sea, and routine monitoring is constantly upgraded. Therefore, scientific and efficient monitoring programmes are needed. Nitrogen is one of the most serious pollutants in the YRE. Obtaining the precise pollution areas of water quality grades (WQGs) are a scientific and management issue that requires optimization of monitoring programmes and interpolation methods. Based on spatiotemporal regression point means of surface with non-homogeneity (STR-PMSN), dissolved inorganic nitrogen (DIN) concentrations were estimated in a stratified heterogeneous estuary. The annual average areas of DIN Grades I and II were classified by interpolating the concentrations; the values were 3145 km2, 1626 km2, 2320 km2 and 3758 km2 for February, May, August and November, respectively. This means that November had the best water condition, and May had the worst. Meanwhile, DIN area changes showed that the water condition changed due to removal of data much more in August and May than in February and November. The descending order of importance was August, May, February and November. Every month represented different runoff periods. Monitoring frequency should not be reduced. Removal of sampling data for the third stratum had a significant effect on the area. When the sampling data for outer boundary meshes of the third stratum were removed, the water condition became worse. However, when the sampling data for inner boundary meshes were removed, the water condition improved. New sites should be added to the outer boundary region to avoid interpolation instability and reduce the sensitivity of the existing sites. This study assesses the spatiotemporal effect of the marine environmental monitoring programmes on pollutant distribution by STR-PMSN, and it offers guidance for more precise data acquisition and processing methods in the YRE and its adjacent sea.

Proteomics analysis of G protein-coupled receptor kinase 4-inhibited cellular growth of HEK293 cells.

G protein-coupled receptor kinases (GRKs) are involved in a wide range of cellular physiology and pathological activities by specifically phosphorylating activated G protein-coupled receptors (GPCRs) to terminate GPCR signaling, or through regulating non-GPCR substrates. We recently reported that overexpression of GRK4 halts cell proliferation and induces cellular senescent phenotype in HEK293 cells. In this study, a quantitative proteomic assay was performed to analyze the protein profiles between HEK293 cells expressing and not expressing GRK4. Results revealed 39 upregulated and 59 downregulated differently expressed proteins (DEPs) in a total of 4124 identified proteins. Gene ontology (GO) annotation and functional enrichment revealed that the DEPs were related to metabolic processes regulated by the binding of these RNA/proteins under the biological processes. The Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis showed pathways of cell development, division, proliferation, apoptosis, aging, autophagy, cell death and cell cycle progression are involved in. Immunoblotting validation of expression of six key target proteins, CALM1, STAT3, CDK1, CDK6, TOP2A, and GRK4, which speculatively maintain abnormal activity in the above pathways, was consistent with the results of proteomics analysis. Lastly, a biological phenotype assay confirmed that GRK4 promoted HEK293 cell growth blockage and G1/0 arrest. Taken together, this study identified some novel molecules that involve in GRK4 signaling and provided valuable information for further studying the mechanisms underlying GRK4-induced proliferative inhibition. SIGNIFICANCE: A quantitative proteomic assay was performed in HEK293 cells expressing and not expressing GRK4 39 upregulated and 59 downregulated differently expressed proteins (DEPs)were identified. DEPs involved in pathways of cell development, division, proliferation, apoptosis, aging, autophagy, cell death and cell cycle progression. Biological phenotype assay confirmed that GRK4 prompted HEK293 cell growth blockage and G1/0 arrest.

G protein-coupled receptor kinase 4-induced cellular senescence and its senescence-associated gene expression profiling.

Senescent cells have lost their capacity for proliferation and manifest as irreversibly in cell cycle arrest. Many membrane receptors, including G protein-coupled receptors (GPCRs), initiate a variety of intracellular signaling cascades modulating cell division and potentially play roles in triggering cellular senescence response. GPCR kinases (GRKs) belong to a family of serine/threonine kinases. Although their role in homologous desensitization of activated GPCRs is well established, the involvement of the kinases in cell proliferation is still largely unknown. In this study, we isolated GRK4-GFP expressing HEK293 cells by fluorescence-activated cell sorting (FACS) and found that the ectopic expression of GRK4 halted cell proliferation. Cells expressing GRK4 (GRK4(+)) demonstrated cell cycle G1/G0 phase arrest, accompanied with significant increase of senescence-associated-ß-galactosidase (SA-ß-Gal) activity. Expression profiling analysis of 78 senescence-related genes by qRT-PCR showed a total of 17 genes significantly changed in GRK4(+) cells (≥ 2 fold, p < 0.05). Among these, 9 genes - AKT1, p16INK4, p27KIP1, p19INK4, IGFBP3, MAPK14, PLAU, THBS1, TP73 - were up-regulated, while 8 genes, Cyclin A2, Cyclin D1, CDK2, CDK6, ETS1, NBN, RB1, SIRT1, were down-regulated. The increase in cyclin-dependent kinase inhibitors (p16, p27) and p38 MAPK proteins (MAPK14) was validated by immunoblotting. Neither p53 nor p21Waf1/Cip1 protein was detectable, suggesting no p53 activation in the HEK293 cells. These results unveil a novel function of GRK4 on triggering a p53-independent cellular senescence, which involves an intricate signaling network.

Curcumol triggers apoptosis of p53 mutant triple-negative human breast cancer MDA-MB 231 cells via activation of p73 and PUMA.

Triple-negative breast cancer (TNBC; estrogen receptor-negative, progesterone receptor-negative and Her-2-negative) is often accompanied by a higher frequency of p53 gene mutations. Therefore, TNBC is challenging to treat due to a lack of biological targets and a poor sensitivity to conventional therapies. Curcumol is a monomer composition isolated from the ethanol extracts of Curcuma wenyujin, a Chinese medicinal herb traditionally used as a cancer remedy. Previous studies have revealed that curcumol is able to block proliferation in various human tumor cell lines. However, the underlying mechanisms have yet to be elucidated. The present study aimed to investigate the anticancer effects of curcumol in the human p53 mutant TNBC MDA-MB-231 cell line and its underlying mechanisms. Cell viability and growth were determined by MTT and a mice xenograft model assay, respectively. Cell cycle distribution was examined by flow cytometry. Apoptosis was evaluated by apoptotic morphology analysis with DAPI staining and flow cytometric analysis following Annexin V/propidium iodide staining. The protein expression in cells was evaluated by immunoblotting. Treatment of MDA-MB-231 cells with curcumol resulted in a significant inhibition of cell proliferation in vitro [half maximal inhibitory concentration (IC50)=240.7±85.0 µg/ml for 48 h and IC50=100.2±13.5 µg/ml for 72 h]. Curcumol treatment also resulted in the suppression of xenograft growth in vivo (100 or 200 µg/kg for 21 days), as well as G1 phase arrest and an apoptotic response, which were accompanied by the upregulation of p73 expression and the activation of the expression of p53 upregulated modulator of apoptosis (PUMA) and Bcl-2 antagonistic killer (Bak). No cleavage of poly (ADP-ribose) polymerase was detected. To the best of our knowledge, the present data demonstrate for the first time that curcumol inhibits the growth of MDA-MB-231 cells and triggers p53-independent apoptosis, which may be mediated by the p73-PUMA/Bak signaling pathway. Curcumol may, therefore, be a potential compound for use in the development of novel TNBC therapeutics.

The regulator of G protein signaling (RGS) domain of G protein-coupled receptor kinase 5 (GRK5) regulates plasma membrane localization and function.

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM). Here GRK5/GRK4 chimeras and point mutations in GRK5 identify a short sequence within the regulator of G protein signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. This region of the RGS domain of GRK5 coincides with a region of GRK6 and GRK1 shown to form a hydrophobic dimeric interface (HDI) in crystal structures. Coimmunoprecipitation (coIP) and acceptor photobleaching fluorescence resonance energy transfer assays show that expressed GRK5 self-associates in cells, whereas GRK5-M165E/F166E (GRK5-EE), containing hydrophilic mutations in the HDI region of the RGS domain, displays greatly decreased coIP interactions. Both forcing dimerization of GRK5-EE, via fusion to leucine zipper motifs, and appending an extra C-terminal membrane-binding region to GRK5-EE (GRK5-EE-CT) recover PM localization. In addition, GRK5-EE displays a decreased ability to inhibit PAR1-induced calcium release compared with GRK5 wild type (wt). In contrast, PM-localized GRK5-EE-CaaX (appending a C-terminal prenylation and polybasic motif from K-ras) or GRK5-EE-CT shows comparable ability to GRK5 wt to inhibit PAR1-induced calcium release. The results suggest a novel model in which GRK5 dimerization is important for its plasma membrane localization and function.

[Mechanism study on anti-proliferative effects of curcumol in human hepatocarcinoma HepG2 cells].

OBJECTIVE: To investigate the anti-proliferative effects of curcumol, an herbal extract from curcuma, in human hepatocarcinoma HepG2 cells, and its possible molecular mechanism. METHOD: The effects of curcumol on human hepatocarcinoma cells were assessed in vitro. Proliferation of HepG2 cells treated with various concentration (2.5-10 mg x L(-1)) of curcumol was determined using the MTT assay and the distribution of cell cycle of HepG2 cells was analyzed using the FCM technique. Expression of 14 cell cycle regulation-related genes were assessed by TaqMan real-time polymerase chain reaction (RT-PCR) method and Western blot. RESULT: Curcumol significantly inhibited the proliferation of HepG2 cells and induced G1 phase arrest in a dose- and time-dependent manner. The mRNA levels of pRB1, cyclin D1, CDK2, CDK8 and p27KIP1 were elevated, while cyclin A1 decreased, in both of the low (25 mg x L(-1)) and the high dose (100 mg x L(-1)) treatment of curcumol. There were no significant changes in the expression of either cyclin E1 or CDK4. The expression of p53 and its target genes p21WAF1 and Wip1 protein were increased. CONCLUSION: Curcumol can inhibit the proliferation of HepG2 cells in vitro and induce G1 arrest of cell cycle through mechanisms activating p53 and pRB pathways that involve genes of cyclin A1, CDK2, CDK8, p21WAF1 and p27KIP1.

A strategy to study pathway cross-talks of cells under repetitive exposure to stimuli.

BACKGROUND: Cells are subject to fluctuating and multiple stimuli in their natural environment. The signaling pathways often crosstalk to each other and give rise to complex nonlinear dynamics. Specifically repetitive exposure of a cell to a same stimulus sometime leads to augmented cellular responses. Examples are amplified proinflammatory responses of innate immune cells pretreated with a sub-threshold then a high dose of endotoxin or cytokine stimulation. This phenomenon, called priming effect in the literature, has important pathological and clinical significances. RESULTS: In a previous study, we enumerated possible mechanisms for priming using a three-node network model. The analysis uncovered three mechanisms. Based on the results, in this work we developed a straightforward procedure to identify molecular candidates contributing to the priming effect and the corresponding mechanisms. The procedure involves time course measurements, e.g., gene expression levels, or protein activities under low, high, and low + high dose of stimulant, then computational analysis of the dynamics patterns, and identification of functional roles in the context of the regulatory network. We applied the procedure to a set of published microarray data on interferon-γ-mediated priming effect of human macrophages. The analysis identified a number of network motifs possibly contributing to Interferon-γ priming. A further detailed mathematical model analysis further reveals how combination of different mechanisms leads to the priming effect. CONCLUSIONS: One may perform systematic screening using the proposed procedure combining with high throughput measurements, at both transcriptome and proteome levels. It is applicable to various priming phenomena.

Downregulation of Wip-1 phosphatase expression in MCF-7 breast cancer cells enhances doxorubicin-induced apoptosis through p53-mediated transcriptional activation of Bax.

The human breast cancer cell line MCF-7 carries an amplified PPM1 D/Wip-1 gene and over expresses Wip-1 phosphatase protein. MCF-7 cells also harbor a wild type p53 gene. We established stable isogenic lines (MCF-Sp53 clones) which exhibit decreased levels of p53 protein. We show that although the PPM1 D gene is amplified in MCF-7 cells it is still expressed in a p53-dependent manner. Stable isogenic cell lines derived from MCF-7 cells (designated MCF-clones) were also established in which Wip-1 expression is significantly decreased by a plasmid-based PPM1D antisense RNA. Decreasing Wip-1 expression sensitized MCF-clones to doxorubicin-induced apoptosis. The enhanced apoptotic response was correlated with increased phosphorylation of N-terrninal p53-Ser15 and -Ser46 and increased expression of the pro-apoptotic Bax gene at both the mRNA and protein level. The enhanced apoptotic response was blocked by Bax-siRNA knock down suggesting that the increased response was a result of increased Bax protein expression. Moreover, reporter gene assays using the Waf-1 and Bax promoters to drive a luciferase gene revealed that luciferase activity driven by the Bax promoter was enhanced in MCF-clones while luciferase activity driven by the Waf-1 promoter was decreased relative to parental MCF-7 cells. The study reveals a novel molecular mechanism involving Wip-1 phosphatase, p53 phosphorylation and an enhanced apoptotic response mediated by transcriptional activation of the pro-apoptotic Bax gene.   W. Edward Mercer Ph.D., who made great contributions to this paper, passed away on Thursday, October 30, 2008, after a brief illness. This paper is in memorial to his honorable attitude toward science and education.

Plasma membrane and nuclear localization of G protein coupled receptor kinase 6A.

G protein-coupled receptor (GPCR) kinases (GRKs) specifically phosphorylate agonist-occupied GPCRs at the inner surface of the plasma membrane (PM), leading to receptor desensitization. Here we show that the C-terminal 30 amino acids of GRK6A contain multiple elements that either promote or inhibit PM localization. Disruption of palmitoylation by individual mutation of cysteine 561, 562, or 565 or treatment of cells with 2-bromopalmitate shifts GRK6A from the PM to both the cytoplasm and nucleus. Likewise, disruption of the hydrophobic nature of a predicted amphipathic helix by mutation of two leucines to alanines at positions 551 and 552 causes a loss of PM localization. Moreover, acidic amino acids in the C-terminus appear to negatively regulate PM localization; mutational replacement of several acidic residues with neutral or basic residues rescues PM localization of a palmitoylation-defective GRK6A. Last, we characterize the novel nuclear localization, showing that nuclear export of nonpalmitoylated GRK6A is sensitive to leptomycin B and that GRK6A contains a potential nuclear localization signal. Our results suggest that the C-terminus of GRK6A contains a novel electrostatic palmitoyl switch in which acidic residues weaken the membrane-binding strength of the amphipathic helix, thus allowing changes in palmitoylation to regulate PM versus cytoplasmic/nuclear localization.

JE Nakayama/JE SA14-14-2 virus structural region intertypic viruses: biological properties in the mouse model of neuroinvasive disease.

A molecular clone of Japanese encephalitis (JE) virus Nakayama strain was used to create intertypic viruses containing either the 5'-C-prM-E or the prM-E region of the attenuated JE SA14-14-2 virus in the JE Nakayama background. These two intertypic JE viruses, JE-X/5'CprME(S) and JE-X/prME(S), respectively, generally resembled the parental JE virus in cell culture properties. Similar to virus derived from the JE Nakayama molecular clone (JE-XJN), JE-X/prME(S) was highly neuroinvasive and neurovirulent for young adult mice, whereas JE-X/5'CprME(S) was attenuated for neuroinvasiveness and only partially attenuated for neurovirulence. Immunization of young mice with JE-X/5'CprME(S) virus elicited neutralizing antibodies against JE Nakayama virus and conferred protection against encephalitis following challenge with JE Nakayama virus. The sequence of the JE-X/5'CprME(S) virus differed from that of JE-X/prME(S) virus at two nucleotides in the 5' UTR, 3 amino acid positions in the capsid protein, 4 positions in the prM protein and 1 in the envelope protein. For JE-X/prME(S) virus, the 4 differences in prM and the single substitution in the envelope represented reversions to the sequence of JE Nakayama virus. Overall, this study reveals that molecular determinants associated with the prM-E region of the attenuated JE SA14-14-2 virus are insufficient by themselves to confer an attenuation phenotype upon JE Nakayama virus. This suggests a role for determinants in the 5' UTR and/or the capsid protein of the JE SA 14-14-2 virus genome in influencing the virulence properties of the JE Nakayama virus in the mouse model.

Chimeric Japanese encephalitis virus/dengue 2 virus infectious clone: biological properties, immunogenicity and protection against dengue encephalitis in mice.

A molecular clone of Japanese encephalitis virus (JE virus) was derived from the JE virus Nakayama strain and used to produce infectious JE virus in cell culture. The engineered JE virus resembled the parental JE virus in cell-culture properties and was related closely to other JE virus strains based on nucleotide sequence analysis. The JE virus clone was used as a genetic background for construction of a chimeric virus containing the structural proteins prM and E of Dengue virus, serotype 2. The chimeric JE/dengue 2 virus generated authentic dengue 2 structural proteins as assessed by immunoassays for the dengue E protein. It exhibited a small plaque size and less efficient growth in various cell lines than the parental JE virus. JE/dengue 2 virus was non-neuroinvasive for young adult mice, but displayed partial neurovirulence at doses up to 4 log p.f.u. given intracerebrally. Immunization of 3-week-old mice with JE/dengue 2 virus yielded neutralizing-antibody titres against dengue 2 virus and conferred protection against dengue encephalitis caused by neuroadapted dengue 2 virus. A rise in post-challenge neutralizing-antibody titres against dengue 2 virus in surviving mice suggests that immunization is associated with establishment of a memory antibody response in this model. This study demonstrates the capacity of JE virus to serve as a vector for expression of heterologous flavivirus structural proteins. Similar to previous studies with other chimeric flaviviruses, this approach may be useful as a genetic system for engineering experimental vaccines against Dengue virus and other medically important flaviviruses.

Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization, immunogenicity, and protection against dengue encephalitis in the mouse model.

Two yellow fever virus (YFV)/dengue virus chimeras which encode the prM and E proteins of either dengue virus serotype 2 (dengue-2 virus) or dengue-4 virus within the genome of the YFV 17D strain (YF5.2iv infectious clone) were constructed and characterized for their properties in cell culture and as experimental vaccines in mice. The prM and E proteins appeared to be properly processed and glycosylated, and in plaque reduction neutralization tests and other assays of antigenic specificity, the E proteins exhibited profiles which resembled those of the homologous dengue virus serotypes. Both chimeric viruses replicated in cell lines of vertebrate and mosquito origin to levels comparable to those of homologous dengue viruses but less efficiently than the YF5.2iv parent. YFV/dengue-4 virus, but not YFV/dengue-2 virus, was neurovirulent for 3-week-old mice by intracerebral inoculation; however, both viruses were attenuated when administered by the intraperitoneal route in mice of that age. Single-dose inoculation of either chimeric virus at a dose of 10(5) PFU by the intraperitoneal route induced detectable levels of neutralizing antibodies against the homologous dengue virus strains. Mice which had been immunized in this manner were fully protected from challenge with homologous neurovirulent dengue viruses by intracerebral inoculation compared to unimmunized mice. Protection was associated with significant increases in geometric mean titers of neutralizing antibody compared to those for unimmunized mice. These data indicate that YFV/dengue virus chimeras elicit antibodies which represent protective memory responses in the mouse model of dengue encephalitis. The levels of neurovirulence and immunogenicity of the chimeric viruses in mice correlate with the degree of adaptation of the dengue virus strain to mice. This study supports ongoing investigations concerning the use of this technology for development of a live attenuated viral vaccine against dengue viruses.

Alternately spliced luteinizing hormone/human chorionic gonadotropin receptor mRNA in human breast epithelial cells.

Luteinizing hormone (LH)/human chorionic gonadotropin (hCG) bind to a common transmembrane glycoprotein receptor, which is a member of the G protein-coupled receptor family. In human, the LH/hCG receptor gene is composed of 11 exons and 10 introns and its coding region is over 60 kb long. Human chorionic gonadotropin is a glycoprotein hormone containing a cystine-knot folding motif that is found in peptide growth factors known to activate the expression of homeogenes. In the present work we present evidence that hCG down-regulates all the three transcripts of HOXA1 at early stages of hCG treatment in the immortalized human breast epithelial cells (MCF-10F), whereas HOXA1-S1, the largest transcript, as well as HOXA1-S3, the smallest transcript, were up-regulated in the cancer cell lines MDA-MB-231 and MCF-7, respectively. This divergent reaction of hCG was associated with the pattern of LH/hCG receptors and splicing forms expression in human breast epithelial cells. MCF-10F cells expressed the full-length (1191 bp) compared with the cancer-derived cells MCF-7 and MDA-MB-231 that was weakly or not expressed. Isoform 1 (1117 bp) was silent in MCF-10F and expressed weakly in the cancer cells. The isoforms 2 (1006 bp) and 3 (932 bp) of LH/hCG gene receptor were silent in all the cell lines, whereas isoforms 4 (892 bp), 6 (626 bp) and 7 (441 bp) were silent in MCF-10F cells and expressed in the cancer cell lines. Instead isoform 5 (707 bp) showed in the three cell lines the strongest expression in MCF-10F cells. This difference in the expression of alternate splicing of LH/hCG receptor mRNA among the MCF-10F, MCF-7 and MDA-MB-231 cells, may explain the divergent response of these cells to HOXA1 activation by hCG.

Human chorionic gonadotropin and inhibin induce histone acetylation in human breast epithelial cells.

We have previously demonstrated that human chorionic gonadotropin or hCG-induces differentiation of the mammary gland. The effect of hCG was accompanied by the synthesis of inhibin, a heterodimeric protein that is structurally related to the transforming growth factor-beta (TGF-beta) family and activation of c-myc, c-jun, testosterone repressed prostate message 2 (TRPM2) and interleukin-l-beta-converting enzyme (ICE) transcripts. In the present work we aim to demonstrate that hCG and inhibin may control the transcription of these genes by acetylation of histones in the breast epithelial cells. For this purpose we have utilized MCF-10F cells in culture to detect the levels of acetylated histone H3 and H4 after treatment with different concentrations of hCG as well as inhibin beta-subunit at various time points. In the Western blot analysis, both acetylated histone H3 and H4 were significantly increased by hCG treatment over 12 h in MCF-10F cells at all the doses tested. Inhibin induced the accumulation of acetylated histone H3 after 4-h-treatment at the concentration of 1 ng/ml and at all the time points with higher concentration (10-1000 ng/ml). Slight induction of acetylated histone H4 was detected only in the cells treated with inhibin at 100 ng/ml for 12 and 24 h. This study is the first one to demonstrate that these hormones increase the acetylation of histones.