Actinidia chinensis Planch Root extract suppresses the growth and metastasis of hypopharyngeal carcinoma by inhibiting E2F Transcription Factor 1-mediated MNX1 antisense RNA 1.

Increasing evidence has shown that traditional Chinese medicines and their bioactive components exert an anti-tumor effect, representing a novel treatment strategy. Actinidia chinensis Planch Root extracts (acRoots) have been reported to repress cancer cell proliferation and metastasis. The effect of acRoots on hypopharyngeal carcinoma progression was explored in this study. Firstly, data from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and colony formation assays showed that incubation with accRoots reduced cell proliferation of hypopharyngeal carcinoma cells. Moreover, acRoots promoted the cell apoptosis of hypopharyngeal carcinoma. Secondly, cell migration and invasion of hypopharyngeal carcinoma cells were suppressed by acRoots. Thirdly, E2F1 (E2F Transcription Factor 1) and lncRNA MNX1-AS1 (MNX1 antisense RNA 1) were up-regulated in hypopharyngeal carcinoma tissues, and reduced in hypopharyngeal carcinoma cells post acRoots incubation. Overexpression of E2F1 attenuated acRoots-induced decrease in MNX1-AS1 in hypopharyngeal carcinoma cells. Lastly, administration with acRoots retarded in vivo hypopharyngeal carcinoma growth through down-regulation of E2F1-mediated MNX1-AS1. In conclusion, acRoots exerted tumor-suppressive role in hypopharyngeal carcinoma through inhibition of E2F1-mediated MNX1-AS1.

Development of a cell-based pathway modulator screening system to screen the targeted cancer therapeutic candidates.

To overcome the issues of poor prognosis and to tackle the non-responsiveness to various chemotherapeutics; it is necessary to develop targeted cancer therapeutic agents. Also, it is being necessary to understand the molecular targets of the drug candidates and drugs in the context of cellular signaling pathways, to make progress towards the development of targeted cancer therapeutics. Towards addressing these, we have established a cell-based and pathway-focused drug screening system for the pathways such as MYC, E2F, WNT, ERK, NRF1/2, HIF1α, p53, YY1 and NFκB. These signaling pathways are highly dysregulated in many cancers, including gastric cancer. The developed firefly luciferase assay-based screening system in gastric cancer lineage is suitable for the screening of the massive panel of drugs, drug candidates, small molecule inhibitors, chemicals and alternate drug formulations. The developed stable cell lines have been demonstrated for their pathway activity reporting features using the corresponding pathway-specific modulators. A proof-of-concept medium throughput screening focusing on YY1 signaling pathway also revealed the connection between calcium channel blockers and YY1 signaling. The developed signaling pathway screening assay cells are valuable resource and will serve as the screening platform for screening the drug libraries towards the development of targeted cancer therapeutics.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

Involvement of pRb-E2F pathway in green tea extract-induced growth inhibition of human myeloid leukemia cells.

Both inhibitory and stimulatory effect of EGCG on cancer cells have been reported, which often is linked to receptor tyrosine kinase signaling. In this study, we present evidence that green tea extract and its chemical component, Epigallocatechin-3-gallate (EGCG), inhibit growth of human myeloid leukemia cells through the regulation of pRb synthesis and formation of pRb-E2F complexes. Addition of green tea extract to the culture of TF-1a and MV4-11 myeloid leukemia cells significantly inhibited their proliferation with a substantial portion of cell death being observed. The green tea extract and EGCG had no significant effect on the expression of G1 CDKs and the CDK inhibitors but downregulated the formation of pRb-CDKs. Surprisingly, the expression of pRb was markedly upregulated while the phosphorylation of pRb downregulated. The upregulation of pRb was blocked by pre-treatment with cycloheximide, a protein synthesis inhibitor, suggesting a requirement of protein synthesis. In agreement with these results, pRb-E2F complexes were upregulated and E2F DNA binding activity decreased. Since both TF-1a and MV4-11 are factor-independent cell lines, the upregulation of pRb-E2F complexes and inhibition of DNA binding activity by green tea extract is most likely through a receptor tyrosine kinase-independent pathway. We also found that the stem/progenitor cells derived from these two leukemia cell lines are more sensitive to the inhibitory effect of green tea extract. Our result suggests that concentrated green tea extract and EGCG may have potential for clinical investigation as an inducer of cancer cell death.

Nascent RNA sequencing reveals a dynamic global transcriptional response at genes and enhancers to the natural medicinal compound celastrol.

Most studies of responses to transcriptional stimuli measure changes in cellular mRNA concentrations. By sequencing nascent RNA instead, it is possible to detect changes in transcription in minutes rather than hours and thereby distinguish primary from secondary responses to regulatory signals. Here, we describe the use of PRO-seq to characterize the immediate transcriptional response in human cells to celastrol, a compound derived from traditional Chinese medicine that has potent anti-inflammatory, tumor-inhibitory, and obesity-controlling effects. Celastrol is known to elicit a cellular stress response resembling the response to heat shock, but the transcriptional basis of this response remains unclear. Our analysis of PRO-seq data for K562 cells reveals dramatic transcriptional effects soon after celastrol treatment at a broad collection of both coding and noncoding transcription units. This transcriptional response occurred in two major waves, one within 10 min, and a second 40-60 min after treatment. Transcriptional activity was generally repressed by celastrol, but one distinct group of genes, enriched for roles in the heat shock response, displayed strong activation. Using a regression approach, we identified key transcription factors that appear to drive these transcriptional responses, including members of the E2F and RFX families. We also found sequence-based evidence that particular transcription factors drive the activation of enhancers. We observed increased polymerase pausing at both genes and enhancers, suggesting that pause release may be widely inhibited during the celastrol response. Our study demonstrates that a careful analysis of PRO-seq time-course data can disentangle key aspects of a complex transcriptional response, and it provides new insights into the activity of a powerful pharmacological agent.

A Strategy for Direct Chemical Activation of the Retinoblastoma Protein.

The retinoblastoma (Rb) tumor suppressor protein negatively regulates cell proliferation by binding and inhibiting E2F transcription factors. Rb inactivation occurs in cancer cells upon cyclin-dependent kinase (Cdk) phosphorylation, which induces E2F release and activation of cell cycle genes. We present a strategy for activating phosphorylated Rb with molecules that bind Rb directly and enhance affinity for E2F. We developed a fluorescence polarization assay that can detect the effect of exogenous compounds on modulating affinity of Rb for the E2F transactivation domain. We found that a peptide capable of disrupting the compact inactive Rb conformation increases affinity of the repressive Rb-E2F complex. Our results demonstrate the feasibility of discovering novel molecules that target the cell cycle and proliferation through directly targeting Rb rather than upstream kinase activity.

Transcription addiction: can we garner the Yin and Yang functions of E2F1 for cancer therapy?

Classically, as a transcription factor family, the E2Fs are known to regulate the expression of various genes whose products are involved in a multitude of biological functions, many of which are deregulated in diseases including cancers. E2F is deregulated and hyperactive in most human cancers with context dependent, dichotomous and contradictory roles in almost all cancers. Cancer cells have an insatiable demand for transcription to ensure that gene products are available to sustain various biological processes that support their rapid growth and survival. In this context, cutting-off hyperactivity of transcription factors that support transcription dependence could be a valuable therapeutic strategy. However, one of the greatest challenges of targeting a transcription factor is the global effects on non-cancerous cells given that they control cellular functions in general. Recently, there is growing realization regarding the possibility to target the oncogenic activation of transcription factors to modulate transcription addiction without affecting the normal activity required for cell functions. In this review, we used E2F1 as a prototype transcription factor to address transcription factor activity in cancer cell functions. We focused on melanoma considering that E2F1 executes critical functions in response to UV, an etiological factor of cutaneous melanoma and lies immediately downstream of the CDKN2A/pRb axis, which is frequently deregulated in melanoma. Further, activation of E2F1 in melanomas can also occur independent of loss of CDKN2A. Given its activated status and the ability to transcriptionally control a plethora of genes involved in regulating melanoma development and progression, we review the current literature on its differential role in controlling signaling pathways involved in melanoma as well as therapeutic resistance, and discuss the practical value of weaning melanoma cells from E2F1-mediated transcription dependence for melanoma management.

Altered invertase activities of symptomatic tissues on Beet severe curly top virus (BSCTV) infected Arabidopsis thaliana.

Arabidopsis thaliana infected with Beet severe curly top virus (BSCTV) exhibits systemic symptoms such as stunting of plant growth, callus induction on shoot tips, and curling of leaves and shoot tips. The regulation of sucrose metabolism is essential for obtaining the energy required for viral replication and the development of symptoms in BSCTV-infected A. thaliana. We evaluated the changed transcript level and enzyme activity of invertases in the inflorescence stems of BSCTV-infected A. thaliana. These results were consistent with the increased pattern of ribulose-1,5-bisphosphate carboxylase/oxygenase activity and photosynthetic pigment concentration in virus-infected plants to supply more energy for BSCTV multiplication. The altered gene expression of invertases during symptom development was functionally correlated with the differential expression patterns of D-type cyclins, E2F isoforms, and invertase-related genes. Taken together, our results indicate that sucrose sensing by BSCTV infection may regulate the expression of sucrose metabolism and result in the subsequent development of viral symptoms in relation with activation of cell cycle regulation.

Galbanic acid decreases androgen receptor abundance and signaling and induces G1 arrest in prostate cancer cells.

Androgen receptor (AR) signaling is crucial for the genesis and progression of prostate cancer (PCa). We compared the growth responses of AR(+) LNCaP and LNCaP C4-2 vs. AR(-) DU145 and PC-3 PCa cell lines to galbanic acid (GBA) isolated from the resin of medicinal herb Ferula assafoetida and assessed their connection to AR signaling and cell cycle regulatory pathways. Our results showed that GBA preferentially suppressed AR(+) PCa cell growth than AR(-) PCa cells. GBA induced a caspase-mediated apoptosis that was attenuated by a general caspase inhibitor. Subapoptotic GBA downregulated AR protein in LNCaP cells primarily through promoting its proteasomal degradation, and inhibited AR-dependent transcription without affecting AR nuclear translocation. Whereas docking simulations predicted binding of GBA to the AR ligand binding domain with similarities and differences with the AR antagonist drug bicalutamide (Bic), LNCaP cell culture assays did not detect agonist activity of GBA. GBA and Bic exerted greater than additive inhibitory effect on cell growth when used together. Subapoptotic GBA induced G(1) arrest associated with an inhibition of cyclin/CDK4/6 pathway, especially cyclin D(1) without the causal involvement of cyclin-dependent kinase (CDK) inhibitory proteins P21(Cip1) and P27(Kip1) . In summary, the novelty of GBA as an anti-AR compound resides in the distinction between GBA and Bic with respect to AR protein turnover and a lack of agonist effect. Our observations of anti-AR and cell cycle arrest actions plus the anti-angiogenesis effect reported elsewhere suggest GBA as a multitargeting drug candidate for the prevention and therapy of PCa.

Growth inhibition of malignant glioblastoma by DING protein.

Malignant gliomas are a highly aggressive type of brain tumor with extremely poor prognosis. These tumors are highly invasive and are often surgically incurable and resistant to chemotherapeutics and radiotherapy. Thus, novel therapies that target pathways involved in growth and survival of the tumor cells are required for the treatment of this class of brain tumors. Previous studies revealed that epidermal growth factor receptor and extracellular-signal-regulated kinases (ERKs), which are involved in the induction of cell proliferation, are activated in the most aggressive type of glioma, i.e. glioblastoma multiforme (GBM). In fact, GBMs with increased levels of ERK activity exhibit a more aggressive phenotype than the others with moderate ERK activity, pointing to the importance of ERK and its kinase activity in the development and progression of these tumors. In this study, we have evaluated the effect of p38SJ, a novel member of the DING family of proteins, derived from Hypericum perforatum calluses, on the growth of malignant glioma cell lines, T98G and U-87MG by focusing on cell cycle and signaling pathways controlled by phosphorylation of various regulatory proteins including ERK. p38SJ, which exhibits profound phosphatase activity, shows the capacity to affect the phosphorylation status of several important kinases modulating signaling pathways, and cell growth and proliferation. Our results demonstrate that p38SJ reduces glioma cell viability and arrests cell cycle progression at G0/G1. The observed growth inhibitory effect of p38SJ is likely mediated by the downregulation of several cell cycle gatekeeper proteins, including cyclin E, Cdc2, and E2F-1. These results suggest that p38SJ may serve as a potential candidate for development of a therapeutic agent for the direct treatment of malignant gliomas and/or as a potential radiosensitizer.

Effects of isotretinoin on spermatogenesis of rats.

BACKGROUND: The results of studies on the effects of retinoids on spermatogenesis are controversial. OBJECTIVES: We evaluated time- and dose-dependent effects of isotretinoin on spermatogenic activity by apoptosis, cyclin D1, E2F, p53 expressions, and Johnsen's scores. METHODS: The rats were divided into three groups. In the 1st group (n = 18), 1 mg/mL/day and in the 2nd group (n = 18) 2 mg/mL/day isotretinoin were administered for 21 days. Flower oil was given to the 3rd (n = 6) control group. On the 7th (groups 1a and 2a), 14th (groups 1b and 2b), and 21st (groups 1c and 2c) days, six rats from the 1st group and six rats from the 2nd group were sacrificed and bilateral orchiectomy was done. RESULTS: The number of cyclin D1 and E2F-positive cells decreased nonsignificantly parallel to days in the 1st group, whereas there was a statistically significant decrease in the 2nd group for the same cells. The p53-positive cells in groups 1c and 2c were increased significantly. CONCLUSIONS: Further studies on the effects of retinoids on spermatogenesis should be conducted. It may be wise to administer contraception to male patients, especially during high-dose and long-term retinoid therapy.

Mammalian target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells in vitro.

The effect of mammalian target of rapamycin (mTOR) inhibitors on pituitary tumors is unknown. Akt overexpression was demonstrated in pituitary adenomas, which may render them sensitive to the anti-proliferative effects of these drugs. The objective of the study was to evaluate the anti-proliferative efficacy of the mTOR inhibitor, rapamycin, and its orally bioavailable analog RAD001 on the GH-secreting pituitary tumor GH3 and MtT/S cells and in human GH-secreting pituitary adenomas (GH-omas) in primary cell cultures. Treatment with rapamycin or RAD001 significantly decreased the number of viable cells and cell proliferation in a dose- and time-dependent manner. This was reflected by decreased phosphorylation levels of the downstream mTOR target p70S6K. Rapamycin treatment of GH3 cells induced G0/G1 cell cycle arrest. In other tumor cell types, this was attributed to a decrease in cyclin D1 levels. However, rapamycin did not affect cyclin D1 protein levels in GH3 cells. By contrast, it decreased cyclin D3 and p21/CIP, which stabilizes cyclin D/cyclin-dependent kinase 4 (cdk4) complexes. Rapamycin inhibited FCS-induced retinoblastoma phosphorylation and subsequent E2F-transcriptional activity. In response to decreased E2F activity, the expression of the E2F-regulated genes cyclin E and cdk2 was reduced. Our results showed that mTOR inhibitors potently inhibit pituitary cell proliferation, suggesting that mTOR inhibition may be a promising anti-proliferative therapy for pituitary adenomas. This therapeutic manipulation may have beneficial effects particularly for patients harboring invasive pituitary tumors resistant to current treatments.

Yin yang 1 modulates taxane response in epithelial ovarian cancer.

Survival of ovarian cancer patients is largely dictated by their response to chemotherapy, which depends on underlying molecular features of the malignancy. We previously identified YIN YANG 1 (YY1) as a gene whose expression is positively correlated with ovarian cancer survival. Herein, we investigated the mechanistic basis of this association. Epigenetic and genetic characteristics of YY1 in serous epithelial ovarian cancer were analyzed along with YY1 mRNA and protein. Patterns of gene expression in primary serous epithelial ovarian cancer and in the NCI60 database were investigated using computational methods. YY1 function and modulation of chemotherapeutic response in vitro was studied using small interfering RNA knockdown. Microarray analysis showed strong positive correlation between expression of YY1 and genes with YY1 and transcription factor E2F binding motifs in ovarian cancer and in the NCI60 cancer cell lines. Clustering of microarray data for these genes revealed that high YY1/E2F3 activity positively correlates with survival of patients treated with the microtubule-stabilizing drug paclitaxel. Increased sensitivity to taxanes, but not to DNA cross-linking platinum agents, was also characteristic of NCI60 cancer cell lines with a high YY1/E2F signature. YY1 knockdown in ovarian cancer cell lines results in inhibition of anchorage-independent growth, motility, and proliferation but also increases resistance to taxanes, with no effect on cisplatin sensitivity. These results, together with the prior demonstration of augmentation of microtubule-related genes by E2F3, suggest that enhanced taxane sensitivity in tumors with high YY1/E2F activity may be mediated by modulation of putative target genes with microtubule function.

Involvement of protein kinase C and E2F-5 in euxanthone-induced neurite differentiation of neuroblastoma.

Euxanthone, a neuritogenic agent isolated from the medicinal herb Polygala caudata, has been shown to induce morphological differentiation and neurite outgrowth in murine neuroblastoma Neuro 2a cells (BU-1 subclone). In order to elucidate the underlying mechanisms of euxanthone-induced neurite outgrowth, a proteomic approach was employed. In the present study, two dimensional (2-D) gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry were performed to investigate the alterations in protein expression profile of euxanthone-treated BU-1 cells. Fourteen identified proteins were changed in expression levels after induction of neurite growth. These proteins included participants in transcription and cell cycle regulation, calcium influx and calcium signaling, fatty acid metabolism, cytoskeleton reorganization, casein kinase signal transduction, putative transbilayer amphipath transport and protein biosynthesis. Among the 14 identified proteins, E2F transcription factor 5 (E2F-5) was significantly up-regulated after euxanthone treatment. Go6976, a protein kinase C (PKC) alpha/betaI inhibitor, was found to inhibit neuritogenesis and expression of E2F-5 in the euxanthone-treated BU-1 cells, while SH-6, the Akt/PKB inhibitor, had no inhibitory effect. The gene silencing of E2F-5 by small interfering RNA (siRNA) was found to abolish the euxanthone-induced neurite outgrowth. In conclusion, these results indicated that the transcription factor E2F-5 was actively involved in the regulation of euxanthone-induced neurite outgrowth via PKC pathway.

HES-1 inhibits 17beta-estradiol and heregulin-beta1-mediated upregulation of E2F-1.

We have previously shown that expression of the transcription factor HES-1 is required for the growth-inhibitory effect of all-trans retinoic acid on MCF-7 cells. In this study, we have used T47D cells with tetracyclin-regulated expression of wild-type or a dominant-negative form of HES-1. Expression of HES-1 in T47D cells inhibited G1/S-phase transition and activation of Cdk2 elicited by estrogen. Estrogen treatment of T47D cells caused increased expression of E2F-1, and this expression was inhibited by cotreatment with all-trans retinoic acid. We show that the effect is mediated through HES-1, which directly downregulates E2F-1 expression through a CACGAG-site within the E2F-1 promoter. Furthermore, proliferation caused by heregulin-beta1 treatment of T47D cells was inhibited by all-trans retinoic acid and this effect was mediated by HES-1. Interestingly, heregulin-beta1-mediated upregulation of E2F-1 expression was directly inhibited by HES-1 through the same CACGAG-site as seen with estrogen-stimulated induction. In addition, we found that two important downstream target genes of estrogen and heregulin-beta1 that are regulated through E2F-1, cyclin E and NPAT, were both regulated in a similar fashion by all-trans retinoic acid, and these effects were antagonized by dominant-negative HES-1. These findings establish that HES-1 inhibits both estrogen- and heregulin-beta1-stimulated growth of breast cancer cells, and further suggest that growth inhibition induced in these cells by all-trans retinoic acid occurs via HES-1-mediated downregulation of E2F-1 expression.

Unique natural antioxidants (NAOs) and derived purified components inhibit cell cycle progression by downregulation of ppRb and E2F in human PC3 prostate cancer cells.

Prostate cancer (PCA) is the leading cause of cancer mortality among older men in Western countries. Epidemiological studies have shown correlation between a lower risk of PCA and a higher consumption of antioxidants. However, the mechanism by which antioxidants exert their effects is still unknown. In the present study, we explored the signaling mechanism through which unique natural antioxidant derived from spinach extract (NAO) exerts their beneficial effects in the chemoprevention of PCA using human PC3 cells. Probing into the effect of NAO and its derived polyphenols on cell-cycle G1 arrest, we found that they cause cell-cycle prolongation. NAO and its two derived purified components exhibited a significant increase in the level of p21cip1 expression after 36 h of starvation, followed by 18 h of treatment with NAO in the presence of serum. In addition, under similar conditions, the expressed level of Cyclin A and CDK-2 in the PC3 cells was significantly reduced after treatment with NAO or its purified components. Immunoblot analysis demonstrated a significant increase in the hypophosphorylated form of pRb and a decrease in ppRb. NAO and its purified derived components were found to downregulate the protein expression of another member of the pRb family, p107, as well as that of E2F-1. These results suggest that NAO-induced G1 delay and cell cycle prolongation are caused by downregulation of the protein expression of ppRb and E2F in the human PCA cell line PC3.

NPDC-1, a novel regulator of neuronal proliferation, is degraded by the ubiquitin/proteasome system through a PEST degradation motif.

Neural proliferation and differentiation control protein-1 (NPDC-1) is a protein expressed primarily in brain and lung and whose expression can be correlated with the regulation of cellular proliferation and differentiation. Embryonic differentiation in brain and lung has classically been linked to retinoid signaling, and we have recently characterized NPDC-1 as a regulator of retinoic acid-mediated events. Regulators of differentiation and development are themselves highly regulated and usually through multiple mechanisms. One such mechanism, protein degradation via the ubiquitin/proteasome degradation pathway, has been linked to the expression of a number of proteins involved in control of proliferation or differentiation, including cyclin D1 and E2F-1. The data presented here demonstrate that NPDC-1 is likewise degraded by the ubiquitin/proteasome system. MG-132, a proteasome inhibitor, stabilized the expression of NPDC-1 and allowed detection of ubiquitinated NPDC-1 in vivo. A PEST motif (rich in proline, glutamine, serine, and threonine) located in the carboxyl terminus of NPDC-1 was shown to target the protein for degradation. Deletion of the PEST motif increased NPDC-1 protein stability and NPDC-1 inhibitory effect on retinoic acid-mediated transcription. NPDC-1 was phosphorylated by several kinases, including extracellular signal-regulated kinase. Phosphorylation of NPDC-1 increased the in vitro rate of NPDC-1 ubiquitination. The MEK inhibitor, PD-98059, an inhibitor of extracellular signal-regulated activation, also inhibited the formation of ubiquitinated NPDC-1 in vivo. Together these results suggest that retinoic acid signaling can be modulated by the presence of NPDC-1 and that the protein level and activity of NPDC-1 can be regulated by phosphorylation-mediated proteasomal degradation.

E2F1 expression is related with the poor survival of lymph node-positive breast cancer patients treated with fluorouracil, doxorubicin and cyclophosphamide.

The expressions of E2F1 and retinoblastoma protein (pRB) were analyzed in 165 lymph node-positive breast cancers. All patients underwent adjuvant chemotherapy with fluorouracil, doxorubicin and cyclophosphamide (FAC). E2F1 was expressed in 43.6% and pRB was expressed in 46.1%. E2F1 expression was significantly increased in pRB-expressing tumors and was associated with an S-phase fraction. By univariate survival analyses, E2F1 expression and ER were identified as significant prognostic factors for disease recurrence and patient survival. E2F1 was the only significant prognostic factor of patient outcome after FAC chemotherapy by multivariate analysis.

Modulation of Sp1-dependent transcription by a cis-acting E2F element in dhfr promoter.

The dihydrofolate reductase (dhfr) promoter contains cis-acting elements for Sp1 and E2F. Here we examined the cooperative regulation of dhfr gene transcription by Sp1 and E2F in human osteosarcoma cells, U2OS. Trichostatin A, an inhibitor of histone deacetylases, markedly stimulated dhfr promoter activity, a response that was enhanced by the deletion of an E2F element. In contrast, deletion of the dhfr Sp1 binding sites completely abolished promoter stimulation by trichostatin A. Cotransfection assays showed that activation of dhfr transcription by expression of E2F1/DP1 requires the reiterated Sp1 elements, whereas activation by Sp1 was enhanced by the deletion of the E2F element. Expression of HDAC1 with Sp1 suppressed promoter activity and suppression was not alleviated by coexpression of E2F1/DP1. These results suggest that HDAC1 acts through Sp1 to repress dhfr promoter activity, and that the E2F element modulates the activity of Sp1 at the dhfr promoter through a cis-acting mechanism.