Understanding the Role of the Transcription Factor Sp1 in Ovarian Cancer: from Theory to Practice.

Ovarian cancer (OC) is one of the deadliest cancers among women contributing to high risk of mortality, mainly owing to delayed detection. There is no specific biomarker for its detection in early stages. However, recent findings show that over-expression of specificity protein 1 (Sp1) is involved in many OC cases. The ubiquitous transcription of Sp1 apparently mediates the maintenance of normal and cancerous biological processes such as cell growth, differentiation, angiogenesis, apoptosis, cellular reprogramming and tumorigenesis. Sp1 exerts its effects on cellular genes containing putative GC-rich Sp1-binding site in their promoters. A better understanding of the mechanisms underlying Sp1 transcription factor (TF) regulation and functions in OC tumorigenesis could help identify novel prognostic markers, to target cancer stem cells (CSCs) by following cellular reprogramming and enable the development of novel therapies for future generations. In this review, we address the structure, function, and biology of Sp1 in normal and cancer cells, underpinning the involvement of Sp1 in OC tumorigenesis. In addition, we have highlighted the influence of Sp1 TF in cellular reprogramming of iPSCs and how it plays a role in controlling CSCs. This review highlights the drugs targeting Sp1 and their action on cancer cells. In conclusion, we predict that research in this direction will be highly beneficial for OC treatment, and chemotherapeutic drugs targeting Sp1 will emerge as a promising therapy for OC.

Diagnostic utility of VEGF mRNA and SP1 mRNA expression in bronchial cells of patients with lung cancer.

BACKGROUND AND OBJECTIVE: Bronchial brushing is important for cytological diagnosis of lung carcinoma; however, cytological evaluation alone remains relatively insensitive. The aim of this study was to assess the diagnostic utility of vascular endothelial growth factor (VEGF) messenger ribonucleic acid (mRNA) and specificity protein 1 (SP1) mRNA in bronchial brushings in patients with or without lung cancer. METHODS: VEGF mRNA and SP1 mRNA were measured in liquid-based cells from bronchial brushings in patients with verified lung cancer (n = 93) and with benign lung disease that included tuberculosis (n = 51). This was done using the reverse-transcription polymerase chain reaction. RESULTS: Both VEGF mRNA and SP1 mRNA were significantly more likely to be expressed in the cancer group than in the control (benign) group, whatever their cell type. It was also more often found in the tuberculosis group than in the inflammation group (P < 0.01). In the cancer group, VEGF mRNA was significantly correlated with SP1 mRNA (P < 0.01). Of the 36 false negative cytology results, 30 gave positive results for VEGF mRNA and 34 for SP1 mRNA. The four false positive VEGF results were all diagnosed as tuberculosis. VEGF mRNA gave the highest diagnostic performance with serial use: sensitivity 89.2% and accuracy 90.3%. This was significantly better than cytology (P < 0.01). CONCLUSIONS: Detection of VEGF mRNA and SP1 mRNA in bronchial brushing cells may be used as an ancillary tool to cytological diagnosis for detection of early-stage lung cancer. It may also help distinguish tuberculosis from other causes of lung inflammation.

Transcription factor-pathway coexpression analysis reveals cooperation between SP1 and ESR1 on dysregulating cell cycle arrest in non-hyperdiploid multiple myeloma.

Multiple myeloma is a hematological cancer of plasma B cells and remains incurable. Two major subtypes of myeloma, hyperdiploid MM (HMM) and non-hyperdiploid MM (NHMM), have distinct chromosomal alterations and different survival outcomes. Transcription factors (TrFs) have been implicated in myeloma oncogenesis, but their dysregulation in myeloma subtypes are less studied. Here, we developed a TrF-pathway coexpression analysis to identify altered coexpression between two sample types. We apply the method to the two myeloma subtypes and the cell cycle arrest pathway, which is significantly differentially expressed between the two subtypes. We find that TrFs MYC, nuclear factor-κB and HOXA9 have significantly lower coexpression with cell cycle arrest in HMM, co-occurring with their overactivation in HMM. In contrast, TrFs ESR1 (estrogen receptor 1), SP1 and E2F1 have significantly lower coexpression with cell cycle arrest in NHMM. SP1 chromatin immunoprecipitation targets are enriched by cell cycle arrest genes. These results motivate a cooperation model of ESR1 and SP1 in regulating cell cycle arrest, and a hypothesis that their overactivation in NHMM disrupts proper regulation of cell cycle arrest. Cotargeting ESR1 and SP1 shows a synergistic effect on inhibiting myeloma proliferation in NHMM cell lines. Therefore, studying TrF-pathway coexpression dysregulation in human cancers facilitates forming novel hypotheses toward clinical utility.

Interleukin-10 genotypes of the -1087 single nucleotide polymorphism influence sp1 expression in periodontitis lesions.

BACKGROUND: Interleukin (IL)-10 is an important cytokine in immune regulation, and the -1087 IL-10 single nucleotide polymorphism (SNP) is associated with chronic periodontitis. The binding of the transcription factor Sp1 to the -1087 position in the IL-10 promoter upregulates IL-10 gene expression, especially in patients with the GG genotype. A correlation between the -1087 GG genotype and high IL-10 and Sp1 gene expressions was found. METHODS: Twenty-five individuals with severe generalized chronic periodontitis were genotyped for the -1087 IL-10 gene polymorphism. SV40 promoter factor 1/specificity protein 1 (Sp1) and IL-10 mRNA were analyzed using a real-time polymerase chain reaction. The amount of Sp1-positive cells and Sp1-positive B cells, as well as the amount of Sp1 protein, in periodontitis lesions were assessed using immunohistochemistry and an in situ proximity ligation assay. RESULTS: The mRNA expression of Sp1 and IL-10 in patients with the GG genotype was four times higher than that in patients with the AA genotype. Proportions of Sp1-positive cells overall and Sp1-positive B cells were larger in patients with the GG genotype than in patients with the AA genotype. CONCLUSION: The transcription factor Sp1 was present in large amounts in periodontitis lesions, and the local expression of Sp1 was related to the -1087 IL-10 SNP.

TLX activates MASH1 for induction of neuronal lineage commitment of adult hippocampal neuroprogenitors.

The orphan nuclear receptor TLX has been proposed to act as a repressor of cell cycle inhibitors to maintain the neural stem cells in an undifferentiated state, and prevents commitment into astrocyte lineages. However, little is known about the mechanism of TLX in neuronal lineage commitment and differentiation. A majority of adult rat hippocampus-derived progenitors (AHPs) cultured in the presence of FGF express a high level of TLX and a fraction of these cells also express the proneural gene MASH1. Upon FGF withdrawal, TLX rapidly decreased, while MASH1 was intensely expressed within 1h, decreasing gradually to disappear at 24h. Adenoviral transduction of TLX in AHP cells in the absence of FGF transiently increased cell proliferation, however, later resulted in neuronal differentiation by inducing MASH1, Neurogenin1, DCX, and MAP2ab. Furthermore, TLX directly targets and activates the MASH1 promoter through interaction with Sp1, recruiting co-activators whereas dismissing the co-repressor HDAC4. Conversely, silencing of TLX in AHPs decreased beta-III tubulin and DCX expression and promoted glial differentiation. Our results thus suggest that TLX not only acts as a repressor of cell cycle and glial differentiation but also activates neuronal lineage commitment in AHPs.

Poor early growth and excessive adult calorie intake independently and additively affect mitogenic signaling and increase mammary tumor susceptibility.

We previously showed that offspring of rat dams receiving a protein-restricted (low protein) diet throughout pregnancy and lactation develop mammary tumors more quickly. Rapid post-weaning mammary growth and mammary tissue overexpression of insulin receptor, insulin-like growth factor-1 receptor (IGF-1R), estrogen receptor isoform alpha and v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), correlated with this risk. The objectives of this study were therefore (i) to identify underlying mechanisms of increased risk through candidate and global approaches; (ii) to determine if excessive calorie intake further increased risk and if so, (iii) to identify the molecular mechanisms mediating this. We provide evidence for transcriptional upregulation of IGF-1R by Sp1 in LP mammary tissue (P < 0.01). Cell cycle control and DNA damage repair gene cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21waf1) was also upregulated (P < 0.05) as was transcription factor nuclear factor of kappa light polypeptide gene enhancer in B-cell (P < 0.05) and adhesion gene CDH1 (P < 0.05). Invasion and metastasis markers matrix metalloproteinase 9 and serpin peptidase inhibitor, clade E, member 1 (SERPIN1) were upregulated (both P < 0.05), whereas metastasis suppressor gene NME1 was downregulated (P < 0.01). Feeding a highly palatable diet (HPD) to increase calorie intake from puberty, additively and independently increased early mammary tumor risk, which correlated with increased serum insulin and triglyceride concentrations (P < 0.05). PTEN gene expression was reduced both by early protein restriction (P < 0.05) and HPD (P < 0.01), which may induce Akt in cell survival pathways. Progesterone receptor and ERBB2 (both P < 0.05) expression increased as an effect of an interaction between maternal diet and adult nutrition, with subsequent downstream activation of the mitogen-activated protein kinase pathway. We conclude that poor early growth and excessive calorie intake exert independent and additive effects on mitogenic growth factor signaling to influence mammary tumor susceptibility.

Mechanisms of decreased expression of transforming growth factor-beta receptor type I at late stages of HPV16-mediated transformation.

Transforming growth factor-beta (TGF-beta) signaling is disrupted in many cancers, including cervical cancer, leading to TGF-beta resistance. Although initially sensitive, human papillomavirus type 16 (HPV16) immortalized human keratinocytes (HKc/HPV16) become increasingly resistant to the growth inhibitory effects of TGF-beta during in vitro progression to a differentiation resistant phenotype (HKc/DR). We have previously shown that loss of TGF-beta sensitivity in HKc/DR is attributed to decreased expression of TGF-beta receptor type I (TGF-beta RI), while the levels of TGF-beta receptor type II (TGF-beta RII) remain unchanged. The present study explored molecular mechanisms leading to reduced TGF-beta RI expression in HKc/DR. Using TGF-beta RI and TGF-beta RII promoter reporter constructs, we determined that acute expression of the HPV16 oncogenes E6 and E7 decreased the promoter activity of TGF-beta RI and TGF-beta RII by about 50%. However, promoter activity of TGF-beta RI is decreased to a greater extent than TGF-beta RII as HKc/HPV16 progress to HKc/DR. Reduced TGF-beta RI expression in HKc/DR was found not to be linked to mutations within the TGF-beta RI promoter or to promoter methylation. Electrophoretic mobility shift and supershift assays using probes encompassing Sp1 binding sites in the TGF-beta RI promoter found no changes between HKc/HPV16 and HKc/DR in binding of the transcription factors Sp1 or Sp3 to the probes. Also, Western blots determined that protein levels of Sp1 and Sp3 remain relatively unchanged between HKc/HPV16 and HKc/DR. Overall, these results demonstrate that mutations in or hypermethylation of the TGF-beta RI promoter, along with altered levels of Sp1 or Sp3, are not responsible for the reduced expression of TGF-beta RI we observe in HKc/DR. Rather the HPV16 oncogenes E6 and E7 themselves exhibit an inhibitory effect on TGF-beta receptor promoter activity.

Up-regulated expression of Sp1 protein coincident with a viral protein in human and mouse differentiating keratinocytes may act as a cell differentiation marker.

Sp1 is a transcription factor that regulates expression of mammalian and viral genes and is involved in different facets of cellular functions in eukaryotic cells. Here, we investigated Sp1 expression in primary mouse and human keratinocyte (KC) culture using quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence microscopy. Expression of Sp1 was post-transcriptionally up-regulated with increasing time in primary KC cultures. Sp1 expression, coincident with expression of human papillomavirus L1 capsid protein and involucrin, was associated with cell differentiation in vitro and in vivo in human and mouse skins. Immunoprecipitation experiments showed that Sp1 and L1 could be bound in a complex. Calcium (Ca(2+)) and all-trans retinoic acid are the positive modulators of KC differentiation, which positively and negatively regulated Sp1 and L1 expression. The data suggest that coincident expression of Sp1 with L1 proteins in differentiating KCs is mediated by a calcium-dependent signaling pathway.

Critical roles of Sp1 in gene expression of human and rat H+/organic cation antiporter MATE1.

A H+/organic cation antiporter (multidrug and toxin extrusion 1: MATE1/SLC47A1) plays important roles in the tubular secretion of various clinically important cationic drugs such as cimetidine. We have recently found that the regulation of this transporter greatly affects the pharmacokinetic properties of cationic drugs in vivo. No information is available about the regulatory mechanisms for the MATE1 gene. In the present study, therefore, we examined the gene regulation of human (h) and rat (r) MATE1, focusing on basal expression. A deletion analysis suggested that the regions spanning -65/-25 and -146/-38 were essential for the basal transcriptional activity of the hMATE1 and rMATE1 promoter, respectively, and that both regions contained putative Sp1-binding sites. Functional involvement of Sp1 was confirmed by Sp1 overexpression, a mutational analysis of Sp1-binding sites, mithramycin A treatment, and an electrophoretic mobility shift assay. Furthermore, we found a single nucleotide polymorphism (SNP) in the promoter region of hMATE1 (G-32A), which belongs to a Sp1-binding site. The allelic frequency of this rSNP was 3.7%, and Sp1-binding and promoter activity were significantly decreased. This is the first study to clarify the transcriptional mechanisms of the MATE1 gene and to identify a SNP affecting the promoter activity of hMATE1.

Association of elevated GRP78 expression with increased lymph node metastasis and poor prognosis in patients with gastric cancer.

Glucose-regulated protein 78 (GRP78) has been implicated in the protection of tumor cells from cytotoxic damage and apoptosis and thus assists cells in survival under oxygen-deprivation and nutrient-stress conditions. However, its expression and potential role in gastric cancer development and progression have not been reported. In the present study, we determined the level of GRP78 expression in the primary tumor in 86 cases of resected gastric cancer by using immunohistochemistry and analyzed the relationships between GRP78 and clinicopathological characteristics. We found that GRP78 was overexpressed in the tumor specimens when compared with the expression in adjacent tumor-free gastric mucosa. Furthermore, the level of GRP78 expression in both primary tumors and metastatic lymph nodes was inversely correlated with patient survival. Overexpression of GRP78 was directly correlated with Sp1 expression and increased lymph node metastasis. Knocking down GRP78 expression inhibited tumor cell invasion in vitro and growth and metastasis in a xenograft nude mouse model. Therefore, our data imply that dysregulated expression of GRP78 may contribute to the development and progression of gastric cancer.

p16 inhibits matrix metalloproteinase-2 expression via suppression of Sp1-mediated gene transcription.

Previous studies demonstrate that p16, a cyclin-dependent kinase inhibitor and a tumor suppressor, may inhibit matrix metalloproteinase-2 (MMP-2) expression in human cancer cells to suppress tumor invasion and metastasis. However, the detailed mechanism is still unclear. Our results show that p16 inhibits MMP-2 expression via transcriptional repression. Promoter deletion and mutation analysis indicates that p16 acts through the Sp1 transcription factor-binding site located between -72 and -64 bp region from the transcriptional start site of the human MMP-2 promoter to repress gene expression. DNA affinity precipitation assay (DAPA) and chromatin immuno-precipitation (CHIP) assay demonstrate that Sp1 proteins constitutively bind to this consensus sequence in vitro and in vivo. p16 attenuates Sp1 binding to the MMP-2 promoter to suppress gene transcription and overexpression of Sp1 may counteract p16-induced downregulation of MMP-2. CyclinA/CDK complex may directly phosphorylate Sp1 and enhance its DNA-binding activity. Thus, we investigated the effect of p16 on the interaction between cyclin A and Sp1. Our results indicate that p16 induces downregulation of cyclin A and CDK2, reduces the interaction between cyclin A and Sp1, and attenuates phosphorylation of Sp1. Ectoexpression of cyclin A counteracts p16-mediated inhibition of DNA binding of Sp1 and activates MMP-2 promoter activity and mRNA expression. Collectively, our results suggest that p16 suppresses MMP-2 by blocking Sp1-mediated gene transcription.

Characterization of the mitochondrial aconitase promoter and the identification of transcription factor binding.

BACKGROUND: Mitochondrial (m) aconitase plays an important role in the unique pathway of citrate accumulation in prostate epithelial cells through its limited activity. In the current study, we characterized the human m-aconitase gene promoter. METHODS: A 1,411-bp 5'-flanking fragment of the human m-aconitase gene was cloned, followed by 5' serial deletion analysis of promoter activity. Transcriptional start sties and transcription factors bound to the promoter were identified by 5' RACE, DNA pull-down assay and transcription factor array analysis. RESULTS: Two transcriptional start sites were identified. The promoter fragment pulled down 15 transcription factors, some without consensus sequences in the promoter. Deletion of one Sp1 site eliminated all promoter activity. CONCLUSIONS: The m-aconitase promoter is contained in a 153-bp 5' fragment lacking a TATA or CAAT sequence. Sp1 binding to a specific Sp1 site is required for promoter activity while other transcription factors are recruited through protein-protein interactions.

Combination analysis of activator protein-1 family members, Sp1 and an activator protein-2alpha-related factor binding to different regions of the urokinase receptor gene in resected colorectal cancers.

PURPOSE: Studies on the transactivation of genes via promoter elements have mostly been done on cell lines rather than resected tissues. This, however, is essential to address an in vivo or clinical relevance. We have previously shown tumor-specific binding of Sp1 and an activator protein (AP)-2-related factor to promoter region -152/-135 of the metastasis-related u-PAR gene in 60% of in vivo-resected cancer tissues. Cell lines have implicated an additional role, and potential synergism, of an AP-1 region (-190/-171) in u-PAR regulation. This study was done to (a) analyze AP-1 binding to this region in resected tumor and normal tissues, and define subgroups in which it is tumor-specific, and (b) to analyze transcription factor-binding patterns to both promoter motifs in resected tissues, supporting synergism, and draw first prognostic conclusions. EXPERIMENTAL DESIGN: In 103 patients with colorectal cancer, electrophoretic mobility shift assay/supershift analysis for u-PAR promoter region -190/-171 was done in tumors and normal tissues. In 71 patients, region -152/-135 was also analyzed. U-PAR protein was measured by ELISA. RESULTS: Tumor-specific AP-1 binding to region -190/-171 of the u-PAR promoter was found in 40% of patients. Subgroup analysis showed tumor-specific binding for c-Fos in 58%, for c-Jun in 50%, for JunD in 39%, and for Fra-1 in 4% of cases. AP-1 binding correlated significantly with u-PAR protein amounts in both normal and tumor tissues (P<0.001), in contrast to a tumor-specific correlation with u-PAR of the AP-2/Sp1 region. In analyses for both promoter regions, 62% of cancers showed simultaneous binding for AP-1, AP-2, and Sp1, 11% for AP-1 and AP-2, 16% for AP-2 and Sp1, 4% for AP-2 only, 3% for AP-1 only, and 0% for Sp1 only. The binding of AP-1, AP-2, and Sp1 correlated significantly with each other (P<0.001), the combination of AP-1 and AP-2 showing the highest correlation with u-PAR (P=0.008). Preliminary survival analysis indicated a trend for poorer prognosis for binding of all three transcription factors. CONCLUSION: This is the first study differentiating transcription factor-binding to two important u-PAR promoter regions in a large series of resected tumors and normal tissues. The AP-1 site seems to be a less tumor-specific regulator than the Sp1/AP-2 motif. Nevertheless, data corroborate the hypothesis of synergism between both elements in resected tumors.

Activation of Sp1-mediated transcription by Rta of Epstein-Barr virus via an interaction with MCAF1.

Rta is a transcription factor encoded by BRLF1 of the Epstein-Barr virus (EBV). This factor is expressed during the immediate-early stage of the lytic cycle to activate the genes required for EBV lytic development. Although transcription activation by Rta is frequently associated with the binding of Rta to the Rta-response element (RRE) in promoters, Rta sometimes activates promoters without an RRE. Here we show that Rta interacts with an Sp1-interacting protein, MBD1-containing chromatin-associated factor 1 (MCAF1). This interaction is critical to the formation of an Sp1-MCAF1-Rta complex at Sp1 sites. Therefore, following lytic induction and the expression of Rta, Rta increases Sp1-mediated transcription. The genes that are thus activated include p16, p21, SNRPN and BRLF1. However, the binding of Rta to RRE prevents the interaction between Rta and MCAF1; therefore, transcription activation by RRE depends only on Rta, and not on MCAF1 or Sp1. Furthermore, this study finds that MCAF1 promotes the expression of Rta and Zta from EBV, indicating that MCAF1 participates EBV lytic activation. Our study documents the critical role of Rta in regulating the transcription of the genes that are mediated by Sp1.

Histopathological and immunohistochemical studies of lenticules after epikeratoplasty for keratoconus.

AIMS: To examine histopathological and immunohistochemical changes in lenticules and host of corneal buttons from patients who previously underwent epikeratoplasty for keratoconus. METHODS: 12 penetrating keratoplasty specimens from patients with keratoconus who had previously undergone epikeratoplasty, eight keratoconus, and seven normal corneas were examined. Immunostaining for Sp1, alpha1-proteinase inhibitor (alpha1-PI), and alpha2-macroglobulin (alpha2M) were performed. RESULTS: In nine of the 12 lenticules, the keratoconus-like disruptions were found in Bowman's layer. Peripheral and posterior keratocyte repopulation of the lenticules was observed in all cases. Keratocyte repopulation in the anterior and mid-stromal regions of the lenticules appeared related to the time since epikeratoplasty. Sp1 nuclear staining of the basal and wing epithelial cells was more intense in lenticules and keratoconus corneas than in normal corneas. Lenticular, host, and keratoconus keratocytes showed positive Sp1 staining, whereas staining was absent in normal corneas. Compared to normal corneas, alpha1-PI and alpha2M immunostaining was lower in the lenticules, host, and keratoconus specimens. CONCLUSIONS: The epithelial cells and keratocytes repopulated in the lenticules retain keratoconus-like biochemical abnormalities such as upregulation of Sp1 and downregulation of alpha1-PI and alpha2M. The authors speculate that both keratocytes and the corneal epithelium may participate in the development of keratoconus.

COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells.

Human immunodeficiency virus type 1 (HIV-1) gene transcription is characterized by two temporally distinct phases. While the initial phase relies solely on cellular transcription factors, the subsequent phase is activated by the viral Tat transactivator. We have previously reported that the subsequent phase of viral gene transcription can be repressed by the chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2 (CTIP2) in human microglial cells [O. Rohr, D. Lecestre, S. Chasserot-Golaz, C. Marban, D. Avram, D. Aunis, M. Leid and E. Schaeffer (2003), J. Virol., 77, 5415-5427]. Here, we demonstrate that CTIP proteins also repress the initial phase of HIV-1 gene transcription, mainly supported by the cellular transcription factors Sp1 and COUP-TF in microglial cells. We report that CTIP2 represses Sp1- and COUP-TF-mediated activation of HIV-1 gene transcription and viral replication as a result of physical interactions with COUP-TF and Sp1 in microglial nuclei. Using laser confocal microscopy CTIP2 was found to colocalize with Sp1, COUP-TF and the heterochromatin-associated protein Hp1alpha, which is mainly detected in transcriptionally repressed heterochromatic region. Moreover, we describe that CTIP2 can be recruited to the HIV-1 promoter via its association with Sp1 bound to the GC-box sequences of the long terminal repeat (LTR). Since our findings demonstrate that CTIP2 interacts with the HIV-1 proximal promoter, it is likely that CTIP2 promotes HIV-1 gene silencing by forcing transcriptionally repressed heterochromatic environment to the viral LTR region.

Analysis of estrogen receptor alpha-Sp1 interactions in breast cancer cells by fluorescence resonance energy transfer.

Estrogen-dependent regulation of several genes associated with cell cycle progression, proliferation, and nucleotide metabolism in breast cancer cells is associated with interactions of estrogen receptor (ER)alpha/Sp1 with GC-rich promoter elements. This study investigates ligand-dependent interactions of ERalpha and Sp1 in MCF-7 breast cancer cells using fluorescence resonance energy transfer (FRET). Chimeric ERalpha and Sp1 proteins fused to cyan fluorescent protein or yellow fluorescent protein were transfected into MCF-7 cells, and a FRET signal was induced after treatment with 17beta-estradiol, 4'-hydroxytamoxifen, or ICI 182,780. Induction of FRET by these ERalpha agonists/antagonists was paralleled by their activation of gene expression in cells transfected with a construct (pSp1(3)) containing three tandem Sp1 binding sites linked to a luciferase reporter gene. In contrast, interactions between ERalpha and Sp1DeltaDBD [a DNA binding domain (DBD) deletion mutant of Sp1] are not observed, and this is consistent with the critical role of the C-terminal DBD of Sp1 for interaction with ERalpha. Results of the FRET assay are consistent with in vitro studies on ERalpha/Sp1 interactions and transactivation, and confirm that ERalpha and Sp1 interact in living breast cancer cells.

Regulation of ceruloplasmin gene in mammals.

A site of rat DNA (about 1800 b. p.) adjacent to the first ceruloplasmin gene contains, apart from regulatory sequences common for all eukaryotic promotors, cis-elements, which are potential binding sites for soluble nuclear receptors of some hormones. Sequences characteristic of genes expressed in liver cells and mammary gland cells during lactation were detected. Full-length fragment of this locus of ceruloplasmin gene (1800 b. p.) was synthesized by PCR and used in gel shift experiments. It was found that soluble proteins extracted from purified nuclei of mammary gland cells during lactation and from the liver of adult and newborn rats, contain proteins specifically interacting with the PCR product. A fragment of chromosome gene containing exons encoding the central part of rat ceruloplasmin was cloned in pTZ19 bacterial vector. Gel shift assay showed that the cloned fragment contained binding sites for specific transcription factor YY1, whose level in nuclear protein fractions varied during ontogeny (according to immunoblotting data). Monoclonal antibodies detected protein YY1 in the complex of cloned DNA-nuclear proteins. Possible mechanisms of tissue-specific regulation of ceruloplasmin gene varying during ontogeny are discussed.

Up-regulation of DNA-dependent protein kinase activity and Sp1 in colorectal cancer.

Tumor tissues and adjacent normal tissues in 12 colorectal cancers were examined for quantitative differences in: i) activity of DNA-dependent protein kinase (DNA-PK), which functions in DNA double-strand breads repair, and ii) protein and mRNA levels of Ku70, Ku80, DNA-PKcs and transcriptional factor Sp1. DNA-PK activity and protein/mRNA levels of Ku70, Ku80, DNA-PKcs and Sp1 were significantly higher in the tumor tissues compared with the normal tissues. Significant correlations between DNA-PK activity and protein/mRNA levels of Ku70, Ku80, DNA-PKcs and Sp1 were observed. Because Ku80 and DNA-PKcs have consensus Sp1 recognition elements in their promoter region, the DNA sequence of Ku70 promoter region was analyzed. Analysis of Ku70 promoter region reveled that Ku70 gene has consensus Sp1 recognition elements in its promoter region. mRNA levels of Ku70, Ku80 and DNA-PKcs were correlated with one another, and significant correlations between Sp1 protein level and mRNA levels of Ku70 and Ku80 were observed. These results suggest that DNA-PK activity and protein- and mRNA-levels of Ku70, Ku80 and DNA-PKcs were elevated in tumor tissues in patients with colorectal cancer because of elevated Sp1 protein levels in tumor tissues.

Transcriptional regulation of P450scc gene expression in the embryonic rodent nervous system.

Steroid hormones are synthesized in adrenals, gonads, placenta, and the central and peripheral nervous systems (neurosteroids). Neurosteroidogenesis, like conventional steroidogenesis, begins with the conversion of cholesterol to pregnenolone, catalyzed by mitochondrial P450 side-chain cleavage enzyme (P450scc). Transcription of the P450scc gene in the adrenals and gonads requires steroidogenic factor-1, which is not expressed in the nervous system cells that express P450scc. A crucial transcriptional regulatory region of the rat P450scc gene is at -130/-94. We have purified two nuclear proteins (70 and 86 kDa) from rat glial C6 cells that specifically bind to the -130/-94 region of the rat P450scc promoter and identified them as the DNA-binding subunits of autoimmune antigen Ku. Ku colocalized with P450scc in several regions of the nervous system, but its overexpression in C6 cells did not augment transcription from a -130/-94 Luciferase construct. Members of the Sp family of transcription factors also bind to the same DNA sequence as Ku. Sp4 and Sp2 colocalize with P450scc in the nervous system early in development, whereas Sp1 and Sp4 colocalize later in development. Sp1 robustly increased transcription from this element in Sp-deficient Drosophila SL2 cells, and Ku synergistically enhanced this Sp1-stimulated transcription. Thus, members of the Sp transcription family play a role in activating P450scc gene transcription in the nervous system, and Ku may further augment this activation.