Identification of circRNAs and circRNA-mRNA network of Epinephelus coioides during Singapore grouper iridovirus infection.

Circular RNA (circRNA), one of the important non-coding RNA molecules with a closed-loop structure, plays a key regulatory role in cell processing. In this study, circRNAs of Epinephelus coioides, an important marine cultured fish in China, were isolated and characterized, and the network of circRNAs and mRNA was explored during Singapore grouper iridovirus (SGIV) infection, one of the most important double stranded DNA virus pathogens of marine fish. 10 g of raw data was obtained by high-throughput sequencing, and 2599 circRNAs were classified. During SGIV infection, 123 and 37 circRNAs occurred differential expression in spleen and spleen cells, indicating that circRNAs would be involved in the viral infection. GO annotation and KEGG demonstrated that circRNAs could target E. coioides genes to regulate cell activity and the activation of immune factors. The results provide some insights into the circRNAs mediated immune regulatory network during bony fish virus infection.

The Role of Epinephelus coioides DUSP5 in Regulating Singapore Grouper Iridovirus Infection.

The dual-specificity phosphatase (DUSP) family plays an important role in response to adverse external factors. In this study, the DUSP5 from Epinephelus coioides, an important marine fish in Southeast Asia and China, was isolated and characterized. As expected, E. coioides DUSP5 contained four conserved domains: a rhodanese homology domain (RHOD); a dual-specificity phosphatase catalytic domain (DSPc); and two regions of low compositional complexity, indicating that E. coioides DUSP5 belongs to the DUSP family. E. coioides DUSP5 mRNA could be detected in all of the examined tissues, and was mainly distributed in the nucleus. Infection with Singapore grouper iridovirus (SGIV), one of the most important pathogens of marine fish, could inhibit the expression of E. coioides DUSP5. The overexpression of DUSP5 could significantly downregulate the expression of the key SGIV genes (MCP, ICP18, VP19, and LITAF), viral titers, the activity of NF-κB and AP-I, and the expression of pro-inflammatory factors (IL-6, IL-8, and TNF-α) of E. coioides, but could upregulate the expressions of caspase3 and p53, as well as SGIV-induced apoptosis. The results demonstrate that E. coioides DUSP5 could inhibit SGIV infection by regulating E. coioides immune-related factors, indicating that DUSP5 might be involved in viral infection.

Selective Antagonism of Lactiplantibacillus plantarum and Pediococcus acidilactici against Vibrio and Aeromonas in the Bacterial Community of Artemia nauplii.

Empiric probiotics are commonly consumed by healthy individuals as a means of disease prevention, pathogen control, etc. However, controversy has existed for a long time regarding the safety and benefits of probiotics. Here, two candidate probiotics, Lactiplantibacillus plantarum and Pediococcus acidilactici, which are antagonistic to Vibrio and Aeromonas species in vitro, were tested on Artemia under in vivo conditions. In the bacterial community of Artemia nauplii, L. plantarum reduced the abundance of the genera Vibrio and Aeromonas and P. acidilactici significantly increased the abundance of Vibrio species in a positive dosage-dependent manner, while higher and lower dosages of P. acidilactici increased and decreased the abundance of the genus Aeromonas, respectively. Based on the liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) analyses of the metabolite of L. plantarum and P. acidilactici, pyruvic acid was used in an in vitro test to explain such selective antagonism; the results showed that pyruvic acid was conducive or suppressive to V. parahaemolyticus and beneficial to A. hydrophila. Collectively, the results of this study demonstrate the selective antagonism of probiotics on the bacterial community composition of aquatic organisms and the associated pathogens. IMPORTANCE Over the last decade, the common preventive method for controlling potential pathogens in aquaculture has been the use of probiotics. However, the mechanisms of probiotics are complicated and mostly undefined. At present, less attention has been paid to the potential risks of probiotic use in aquaculture. Here, we investigated the effects of two candidate probiotics, L. plantarum and P. acidilactici, on the bacterial community of Artemia nauplii and the in vitro interactions between these two candidate probiotics and two pathogens, Vibrio and Aeromonas species. The results demonstrated the selective antagonism of probiotics on the bacterial community composition of an aquatic organism and its associated pathogens. This research contributes to providing a basis and reference for the long-term rational use of probiotics and to reducing the inappropriate use of probiotics in aquaculture.

Analysis of differentially expressed genes in LNCaP prostate cancer progression model.

The LNCaP/C4-2 human prostate cancer progression model was established to mimic phenotypic and genotypic changes during prostate cancer development from androgen dependence to androgen independence, from nonmetastasis to metastasis. In this study, cDNA microarrays were performed using a microarray chip from Affymetrix to characterize and compare gene expression profiles in LNCaP and C4-2, which may provide novel insight into the molecular mechanism mediating prostate cancer progression. Three hundred eighteen genes consistently exhibited differential expression in LNCaP and C4-2 in 2-time microarray data. Based on their function, the differentially expressed genes can be grouped into several subcategories, including growth factors and signal transducers, oncogenes and tumor suppressors, tumor-specific antigens, transcriptional factors, transporters, and factors involved in invasion, metastasis, and metabolism. Some genes are novel and unexplored in prostate cancer progression and are of potential interest for follow-up investigation. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR were performed to corroborate the microarray results, and 76 differentially expressed genes were validated out of 104 candidates. Expression pattern analyses were performed in these 76 differentially expressed genes, and a series of genes was found to be positively or negatively correlated to prostate cancer progression in the LNCaP prostate cancer progression model and to possess predominant prostate cell specificity. ELF5/ESE-2b and long-chain acyl coenzyme A dehydrogenase (ACADL) expressions were found to be positively associated with malignant progression in LNCaP, C4-2, and C4-2B, and predominantly expressed in prostate cancer cells. Functional evaluation revealed that ELF5/ESE-2b and ACADL expressions contributed to the malignant phenotypes of prostate cancer cells. Accordingly, our microarray data may provide clues for finding novel genes involved in prostate cancer progression to androgen independent and metastasis, and shed light on finding new targets for diagnosis and therapy of prostate cancer.

Synergistic repression of estrogen receptor transcriptional activity by FHL2 and Smad4 in breast cancer cells.

Four and a half LIM domain protein 2 (FHL2) has been implicated in development and progression of various types of cancers. However, little is known about the biological function of FHL2 in breast cancer. Here, we report that FHL2 physically and functionally interacts with estrogen receptors (ERα and ERß), important regulators of breast cancer development and progression. The N-terminal half LIM domain or a single LIM domain of FHL2 was sufficient for its interaction with ERα and ERß. Overexpression of FHL2 reduced ER transcriptional activity in breast cancer cells, whereas reduction of endogenous FHL2 with FHL2 small interfering RNA enhanced ER transactivation. Moreover, FHL2 cooperates with Smad4, a previously known corepressor for ERα, to inhibit ERα transcriptional activity as well as expression of the estrogen-responsive gene cathepsin D. The synergistic inhibition of ERα transcriptional activity by FHL2 and Smad4 may be due to enhanced interaction of Smad4 with ERα by FHL2, because FHL2(1-156), the FHL2 deletion mutant, which showed no synergistic effect, failed to increase such interaction. These data suggested the cooperative regulation of estrogen signaling by FHL2 and Smad4 in breast cancer cells, and might provide a new regulation mechanism underlying breast cancer development and progression.

[Molecular mechanisms of regulation of estrogen receptor a expression level in breast cancer].

Estrogen receptor alpha (ERalpha) plays an important role in breast cancer development and progression and thus becomes a useful molecular target for breast cancer therapy. ERalpha is differentially expressed in breast cancer patients. Moreover, ERalpha expression levels may change at different stages of breast cancer even for the same patient. ERalpha expression is closely associated with the effect of endocrine therapy and prognosis. The mechanisms underlying ERalpha expression are complicated, because ERalpha expression is regulated at different levels, including chromatin, transcriptional, post- transcriptional, translational, and post-translational levels. Many proteins modulate the transcription of ERalpha gene at the chromatin and transcriptional levels through direct or indirect interaction with the ERa promoter. Some microRNAs decrease ERalpha levels possibly by induction of the degradation of ERalpha mRNA and/or repression of the mRNA translation. At the post-translational level, many proteins regulate ERalpha protein levels through ubiquitin-proteosome pathway. This review focuses on molecular mechanisms of regulation of ERalpha expression at different levels.

Ubiquitous mitochondrial creatine kinase is overexpressed in the conditioned medium and the extract of LNCaP lineaged androgen independent cell lines and facilitates prostate cancer progression.

BACKGROUND: Androgen independent prostate cancer (AIPC) is not responsive to androgen ablation therapy. The biomarkers of AIPC are lack. Numerous proteomics studies have focused on finding new markers of AIPC and exploring their possible functions, but little is known about the difference between conditioned medium (CM) from AIPC and androgen dependent prostate cancer (ADPC) cells. METHODS: We performed a proteome analysis of CM from LNCaP, C4-2, and C4-2B cells by a two dimensional electrophoresis based technology. Western blots and immunohistochemical studies were employed to explore the expression pattern of the identified protein in prostate cancer cell lines and clinical specimens, respectively. Then we examined the possible roles and mechanisms of the ubiquitous mitochondrial creatine kinase (uMtCK) in vitro. RESULTS: Besides prostate specific antigen (PSA) and insulin-like growth factor binding protein-2 (IGFBP2), uMtCK was identified in the CM of AIPC cells. uMtCK was up-regulated in AIPC cells and in human prostate cancer tissues at WHO grade III. Stably transfected exogenous uMtCK showed a growth promoting effect rather than mock vector in LNCaP cells, with or without bicalutamide in culture medium. Further assays showed that higher degrees of ROS generation and Akt signaling pathway activation in LNCaP-uMtCK than in LNCaP-neo cells. CONCLUSIONS: We showed that uMtCK could be easily detected in CM of LNCaP lineaged AIPC cells. Exogenous uMtCK in LNCaP cells surprisingly contributed to overproduction of ROS, activation of Akt signaling pathway and more aggressive phenotypes including androgen independence development.

Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-beta-like signaling pathway.

The four-and-a-half LIM (FHL) proteins belong to a family of LIM-only proteins that regulate cell proliferation, differentiation, and apoptosis. The exact functions of each FHL protein in cancer development and progression remain unknown. Here we report that FHL1, FHL2, and FHL3 physically and functionally interact with Smad2, Smad3, and Smad4, important regulators of cancer development and progression, in a TGF-beta-independent manner. Casein kinase 1delta, but not the TGF-beta receptor, was required for the FHL-mediated TGF-beta-like responses, including increased phosphorylation of Smad2/3, interaction of Smad2/3 and Smad4, nuclear accumulation of Smad proteins, activation of the tumor suppressor gene p21, and repression of the oncogene c-myc. FHL1-3 inhibited anchorage-dependent and -independent growth of a human hepatoma cell line in vitro and tumor formation in nude mice. Further analysis of clinical samples revealed that FHL proteins are often downregulated in hepatocellular carcinomas and that this correlates with decreased TGF-beta-like responses. By establishing a link between FHL proteins and Smad proteins, this study identifies what we believe to be a novel TGF-beta-like signaling pathway and indicates that FHL proteins may be useful molecular targets for cancer therapy.

High mobility group box-1 protein acts as a coactivator of nuclear factor of activated T cells-2 in promoting interleukin-2 transcription.

High mobility group box-1 protein, an abundant and conserved constituent of vertebrate nuclei, has recently been reported to be an endogenous immune signal [Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, et al. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Reports 2004;5:825-30]. High mobility group box-1 protein can trigger the release of interleukin-2 and interleukin-12 from lymphocytes. However, at present the underlying mechanism remains unknown. It has been clarified that nuclear factor of activated T cells-2 transduces most immunological signals in T cells and modulates the production of interleukin-2. So it is natural that we asked whether high mobility group box-1 protein could promote production of interleukin-2 in a nuclear factor of activated T cells-2-dependent way. Our experiments firstly showed that high mobility group box-1 protein could bind to nuclear factor of activated T cells-2 in vivo and in vitro. High mobility group box-1 protein cotransfection markedly upregulated the transcription activity of nuclear factor of activated T cells-2 in promoting interleukin-2 reporter gene transcription, which was demonstrated to be dose-dependent. Cotransfection of high mobility group box-1 protein and nuclear factor of activated T cells-2 induced an 18.4-time increase of interleukin-2 activity in 293T cells and a 117.7-time increase in Hela cells. Moreover, inhibition of either high mobility group box-1 protein or nuclear factor of activated T cells -2 expression by sRNAi led to significant decrease of transcription activity of interleukin-2 reporter gene, suggesting that high mobility group box-1 protein and nuclear factor of activated T cells-2 both take important roles in facilitating interleukin-2 transcription, and high mobility group box-1 protein could act as a coactivator for nuclear factor of activated T cells-2 in enhancing transcription of interleukin-2. This discovery has not been reported elsewhere, and helps to understand the newly highlighted immunological role of high mobility group box-1 protein.

[Effects of trichostatin A on the interaction between HBx and histone deacetylase protein 1].

OBJECTIVES: To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1). METHODS: Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo. RESULTS: Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1. CONCLUSIONS: HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

[Heterologous genes expression on Escherichia coli chromosome lac operon using Red recombination].

To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.

Nucleolar localization of TERT is unrelated to telomerase function in human cells.

Telomerase maintains telomere length and has been implicated in both aging and carcinogenesis of human cells. This enzyme is a specialized ribonucleoprotein (RNP) complex, minimally consisting of two essential components: the protein catalytic subunit TERT (telomerase reverse transcriptase) and the integral RNA moiety TR (telomerase RNA, TERC). Both TERT and TR have been found to localize to nucleoli within the nucleus, leading to the suggestion of nucleoli as the site for telomerase RNP biogenesis in human cells. However, whether this statement is true or not has not yet been convincingly demonstrated. Here, we identify that residues 965-981 of the human TERT polypeptide constitute an active nucleolar-targeting signal (NTS) essential for mediating human TERT nucleolar localization. Mutational inactivation of this NTS completely disrupted TERT nucleolar translocation in both normal and malignant human cells. Most interestingly, such a TERT mutant still retained the capacity to activate telomerase activity, maintain telomere length and extend the life-span of cellular proliferation, as does wild-type TERT, in BJ cells (normal fibroblasts). Therefore, our data suggest that TERT nucleolar localization is unrelated to telomerase function in human cells.

PC-1/PrLZ contributes to malignant progression in prostate cancer.

PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.

The antibody preparation and expression of human Pescadillo.

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigenesis, the cDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated. Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells, and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer, colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a potential target in cancer diagnosis and therapy.

Identification and characterization of the novel human prostate cancer-specific PC-1 gene promoter.

Human prostate and colon gene-1 (PC-1, also known as PrLZ) is an androgen-regulated, prostate tissue and prostate cancer cells specifically expressed novel gene. The increased expression of PC-1 gene appears to promote prostate cancer cells androgen-dependent (AD) and androgen-independent (AI) growth. To clone and investigate the expression and regulation elements of PC-1 gene may provide insight into the function of PC-1 and develop a new promoter that targets therapeutic genes to the AD and AI prostate cancer cells. The goal of the present study is cloning and characterization of the PC-1 promoter. A series of luciferase constructs that contain various fragments of the PC-1 5'-genomic region were transfected into human prostate cancer cells for promoter transactivation analysis. 5' deletion analysis identified the -1579 bp promoter region was required for the maximal proximal promoter activity; two transcriptional suppression and a positive regulatory region were identified; -4939 bp promoter fragment of the PC-1 gene retained the characteristic of prostate cancer-specific expression and exhibited higher transcription activity than PSA-6 kb promoter in the medium supplemented with steroid-depleted FBS. An androgen response element (ARE) was located in between -345 and -359 bp of the PC-1 5'-untranslated region relative to the translation initiation site. Thus, our studies not only provide molecular basis of PC-1 transcription regulation, but also define a new regulatory sequence that may be used to restrict expression of therapeutic genes to prostate cancer in the prostate cancer gene therapy.

Characterization of a novel effect of hPinX1 on hTERT nucleolar localization.

The factor PinX1 has been shown as a telomerase inhibitor evolutionarily conserved in both the yeast and the human being. yPinX1 inhibits telomerase activity by sequestering yTERT (telomerase reverse transcriptase) from uniting with yTR (telomerase template RNA) in the nucleolus of yeast cells. However, the mechanism underlying the action of hPinX1 on telomerase regulation in human cells is not known. We here demonstrated that hPinX1 actually has an effect on mediating hTERT nucleolar localization and this effect is mediated by a novel domain enclosed within the central section of the polypeptide. Interestingly, we showed that a reported cancerous mutant form of hPinX1, in which residues of Ser254 and Cys265 are, respectively, mutated to Cys and Tyr, lost the activity on mediating hTERT nucleolar localization. Finally, we provided evidence that mediation of hTERT nucleolar localization and telomerase enzymatic inhibition are two separated function of hPinX1 on telomerase regulation in human cells.

Tissue type-specific modulation of ER transcriptional activity by NFAT3.

NFAT3 belongs to the NFAT family of transcription factors playing important roles in the development of several organ systems and was found to act as a transcriptional coactivator of estrogen receptors (ERalpha and ERbeta) in breast cancer cells. Since some cofactors of transcription factors show cell or tissue type-specific effects on transcriptional regulation, we investigated the effect of NFAT3 on the transcriptional activity of ERs in different cell lines originated from kidney. Surprisingly, overexpression of NFAT3 in these cell types decreased dose-dependently both ERalpha and ERbeta transcriptional activities in a ligand-independent manner. Knockdown of endogenous NFAT3 using NFAT3 small interfering RNA (siRNA) increased ER transcriptional activities. NFAT3 deletion mutants lacking the ER-binding sites completely abolished the NFAT3 repression of ERalpha and ERbeta transcriptional activities. Replacement of Ser168 and Ser170, the amino acid residues on which NFAT3 can be phosphorylated, with Ala did not change the ability of NFAT3 to inhibit the transcriptional activity of ERalpha and ERbeta. Taken together, these results demonstrate that NFAT3 is a new kind of cofactor that displays dual transcription modulation mode dependent on tissue types.

[Stress proteins of Bifidobacterium longum NCC2705].

Stress proteins of Bifidobacterium longum strain NCC2705 were identified and characterized during stationary phase. According to the proteomic map of L. lactics IL1403 and theoretical Mr/pl of stress proteins in B. longum NCC2705 genome annotation, spots of stress proteins in gels were localized, and proteins were identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and/or ESI-MS/MS mass spectrometry. For protein identification by peptide mass fingerprinting, peptide masses were searched against database of B. longum NCC2705 by Mascot licensed in-house. 44 spots representing 8 protein entries have been identified, these proteins were hydrophilic proteins and predicted acid proteins. Spot and protein analysis revealed that post-translational modifications might be common in these proteins. Except for DnaJ, the stress proteins were encoded by genes with CAI value above 0.5, and represented a large proportion of the most abundant proteins. Moreover, the results of scavenging effects on free radicals in vitro showed that B. longum NCC2705 can inhibit fatty acid oxidation and scavenge DPPH, but they scavenge weakly active oxygen free radicals. We identified a key protein that can reverse oxidative damage to proteins and lipids: alkyl hydroperoxide reductase (ahpC, BL0615)synthesized under our experimental conditions.

[An analysis of the features of HBx protein distributed in liver cells and its expression in E. coli].

OBJECTIVE: To investigate the features of HBx protein distributed in liver cells and its expression in E. coli. METHODS: The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography. RESULTS: The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded. CONCLUSION: This study may provide a basis for further study on the biological function of HBx at the protein level.

[Effects of exogenous ER beta expression on the cell growth properties of MCF-7 breast cancer cell line].

OBJECTIVE: To study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment. METHODS: An eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed. RESULTS: A stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed. CONCLUSION: Exogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.