LncRNA CCAT1 promotes the progression of preeclampsia by regulating CDK4.

OBJECTIVE: Preeclampsia is one of the leading causes of maternal and perinatal deaths. This study mainly explored the mechanism of long non-coding RNA (lncRNA) CCAT1 expression in the placenta of preeclampsia patients and its effect on the progression of preeclampsia. MATERIALS AND METHODS: We used quantitative reverse transcription PCR (qRT­PCR) to detect the lncRNA CCAT1 expression in 40 preeclampsia and 40 normal pregnancy placenta samples. CCAT1 expression and its relationship with the clinicopathological parameters of preeclampsia was statistically analyzed. The specific small interfering RNA (si-CCAT1) and plasmid (pcDNA-CCAT1) targeting lncRNA CCAT1 were synthesized and transfected into Bew and JEG-3 cells. The CCAT1 expression in Bew and JEG-3 cells was determined by qRT­PCR. The effect of overexpression and interference of lncRNA CCAT1 on the proliferation of Bew and JEG-3 cells was observed. The effect of CCAT1 on cell cycle was examined by cell cycle assay. The protein expression was accessed by Western blot. RESULTS: Higher lncRNA CCAT1 expression was found in preeclampsia patients. The systolic blood pressure, diastolic blood pressure and urine protein in preeclampsia patients were significantly higher than those in normal pregnant women. The birth weight of fetus was significantly lower than that of normal pregnant women. However, there was no significant difference in weight and age of patients. According to the CCAT1 expression, preeclampsia patients were assigned into high expression group and low expression group. Higher systolic blood pressure, diastolic blood pressure, and urinary protein levels in CCAT1 high expression group were observed comparing to those in low expression group, while the birth weight in low expression group was significantly higher than the high expression group. In addition, we found that after interference with CCAT1, trophoblast proliferation was significantly increased and cell cycle was significantly accelerated, whereas overexpression of CCAT1 led to the contrary. Western blotting indicated that the expressions of E2F1, cyclin D, CDK2 and CDK4 in BeWo cells were increased after CCAT1 was knocked down. The expressions of E2F1, cyclin D, CDK2 and CDK4 in JEG3 cells were decreased after CCAT1 was overexpressed. CONCLUSIONS: LncRNA CCAT1 was highly expressed in preeclampsia and can promote the progression of preeclampsia by inhibiting the expression of CDK4.

E2F1 Signalling is Predictive of Chemoresistance and Lymphogenic Metastasis in Penile Cancer: A Pilot Functional Study Reveals New Prognostic Biomarkers.

BACKGROUND: For penile cancer (PC) there are no known molecular predictors of lymphatic spread and/or chemoresistance. OBJECTIVE: To identify functional biomarkers that can predict malignant progression and treatment responsiveness. DESIGN, SETTING, AND PARTICIPANTS: We used four patient-derived PC cell lines and measured invasion and capillary tube formation, chemoresponsiveness, and mRNA and protein expression. Data were further validated in E2F1 transcription factor knockdown and overexpression experiments. We quantified E2F1 transcript levels in a set of nonmetastatic tumours (NM), metastasised primary tumours (PT), and lymph node metastases (M) from 24 patients. E2F1 immunohistochemistry was performed in another set of 13 PC biopsies. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Relationships between different parameters were analysed using Student t tests. Transcript levels in patient samples were compared using Mann-Whitney U tests. Significance was set at p<0.05. RESULTS AND LIMITATIONS: In cell lines established from lymph node metastases, E2F1 was more abundantly expressed, pRB was inactivated, and CDK2, CDK4, and cyclins D and E were elevated in comparison to cells from primary PC. Overexpression of E2F1 enhanced migratory capacity and lymphatic endothelial tubule formation, while depletion reduced invasiveness and increased chemosensitivity. VEGFR-3 and VEGF-C and mesenchymal markers were upregulated by high E2F1. E2F1 was clearly upregulated in infiltrative and metastatic primary tumours and metastases (NM vs PT, p<0.05; NM vs M, p<0.0005). E2F1 Quick scores increased from grade I to grade III tumours. A limitation of the study is the small number of patients. CONCLUSIONS: E2F1 is a driver of invasion and lymphatic dissemination and promotes chemoresistance. E2F1-related biomarkers might assist in stratifying PC patients for different treatment regimens. PATIENT SUMMARY: The availability of penile cancer cell lines allows molecular research on the mechanisms underlying metastasis and chemotherapy. A critical pathway involved in both features has been identified and may lead to better patient stratification for treatment selection.

Histological and immunohistochemical markers for progression prediction in transurethrally resected high-grade non-muscle invasive bladder cancer.

High-grade non-muscle-invasive bladder cancer (Non-MIBC) has a high risk of stage progression to muscle-invasive bladder cancer (MIBC) and could be managed either conservatively by transurethral resection of bladder tumor (TURBT) or more aggressively by radical cystectomy. The selection of patients who may benefit from early radical intervention is a challenge. To define useful prognostic markers for progression, we analyzed clinicopathological features and immunohistochemical expression patterns of E2F1, p27, survivin, p53, EZH2, IMP3, TSC1/hamartin, fatty acid synthase, androgen receptor, 14-3-3σ, MAGEA4, and NY-ESO-1 on 118 cases of high-grade Non-MIBC. During the mean follow-up period of 64.3 months, progression occurred in 18 patients (15.3%). Histologically, large amount of invasive component (> 50%) was noted in 35 cases (29.7%) and was strongly associated with progression. Among the 12 biomarkers, high expressions of E2F1 and nuclear p27 were noted in 46 cases (40.0%) and 14 cases (12.7%), respectively, and were associated with frequent progression. Using multivariate analysis, the proportion of invasive component and high E2F1 expression were independent prognostic factors for the prediction of progression. Our results indicated that large amount of invasive carcinoma component and high expressions of p27 and E2F1 were predictive markers for progression in Non-MIBC. Therefore, we suggest that these parameters, especially proportion of invasive carcinoma component and E2F1 expression, should be evaluated during pathologic examination and considered during selection of the appropriate management strategy for high grade Non-MIBC patients.

Tudor staphylococcal nuclease (Tudor-SN), a novel regulator facilitating G1/S phase transition, acting as a co-activator of E2F-1 in cell cycle regulation.

Tudor staphylococcal nuclease (Tudor-SN) is a multifunctional protein implicated in a variety of cellular processes. In the present study, we identified Tudor-SN as a novel regulator in cell cycle. Tudor-SN was abundant in proliferating cells whereas barely expressed in terminally differentiated cells. Functional analysis indicated that ectopic overexpression of Tudor-SN promoted the G1/S transition, whereas knockdown of Tudor-SN caused G1 arrest. Moreover, the live-cell time-lapse experiment demonstrated that the cell cycle of MEF(-/-) (knock-out of Tudor-SN in mouse embryonic fibroblasts) was prolonged compared with wild-type MEF(+/+). We noticed that Tudor-SN was constantly expressed in every cell cycle phase, but was highly phosphorylated in the G1/S border. Further study revealed that Tudor-SN was a potential substrate of Cdk2/4/6, supportively, we found the physical interaction of endogenous Tudor-SN with Cdk4/6 in G1 and the G1/S border, and with Cdk2 in the G1/S border and S phase. In addition, roscovitine (Cdk1/2/5 inhibitor) or CINK4 (Cdk4/6 inhibitor) could inhibit the phosphorylation of Tudor-SN, whereas ectopic overexpression of Cdk2/4/6 increased the Tudor-SN phosphorylation. The underlying molecular mechanisms indicated that Tudor-SN could physically interact with E2F-1 in vivo, and could enhance the physical association of E2F-1 with GCN5 (a cofactor of E2F-1, which possesses histone acetyltransferase activity), and promote the binding ability of E2F-1 to the promoter region of its target genes CYCLIN A and E2F-1, and as a result, facilitate the gene transcriptional activation. Taken together, Tudor-SN is identified as a novel co-activator of E2F-1, which could facilitate E2F-1-mediated gene transcriptional activation of target genes, which play essential roles in G1/S transition.

Expressions and clinical significances of c-MET, p-MET and E2f-1 in human gastric carcinoma.

BACKGROUND: To investigate on the expressions and the clinical significances of hepatocyte growth factor receptor (c-MET), phosphorylated c-MET (p-MET) and e2f-1 transcription factor in primary lesion of gastric adenocarcinoma (GC). METHOD: Tissue samples from the primary lesion of GC in patients who accepted D2/D3 radical gastrectomy with R0/R1 resection were stained by immunohistochemistry of c-MET, p-MET, e2f-1 and Ki-67. The univariate and the multivariate analyses involving in clinicopathological parameters and prognostic factors were evaluated. RESULTS: The positivity rates for c-MET (66.12%, 80 cases/121 cases), p-MET (59.50%, 72 cases/121 cases), e2f-1 (38.84%, 47 cases/121 cases) and Ki-67 (72.73%, 88 cases/121 cases) in primary lesion of GC was significantly higher than that in non-cancerous tissue at 5 cm places far from the margin of primary lesion (P < 0.05, respectively). The deeper tumor invasion, the severer lymph node metastasis, the later stage of TNM and the higher expression of Ki-67 was respectively an independent risk factor for the higher expression of c-MET or p-MET, but the younger age and the shorter survival time was an independent risk factor for the higher expression of e2f-1 respectively. Survival analysis showed that the worse prognosis could be observed in the patients with the combination of both c-MET-positive and e2f-1-negative (P = 0.038) or both p-MET-positive and e2f-1-negative (P = 0.042). Cox analysis demonstrated that the severer lymphatic node metastasis and the higher positivity rate of c-MET, p-MET or e2f-1 were an independent prognosis factor respectively. The higher expression of e2f-1 was identified in patients with Stage I-II, which correlated with a shorter survival time. Survival analysis also revealed that the prognosis of patients with positive expression of e2f-1 at Stage I-II was significantly worse than that in patients with negative expression of e2f-1 (χ2 = 13.437, P = 0.001). However, in the cases with Stage III-IV, no significant difference could be identified in the prognostic comparison between positive and negative expressions of e2f-1. CONCLUSIONS: The expression of c-MET or p-MET is an independent prognosis factor. It has been observed that the higher expression of e2f-1 occurred in the early stages while the lower expression of it in the later stages in GC.

E2F4 plays a key role in Burkitt lymphoma tumorigenesis.

Sporadic Burkitt lymphoma (sBL) is a rapidly growing B-cell non-Hodgkin's lymphoma whose treatment requires highly aggressive therapies that often result severely toxic. Identification of proteins whose expression or function is deregulated in sBL and play a role in its formation could facilitate development of less toxic therapies. We have previously shown that E2F1 expression is deregulated in sBL. We have now investigated the mechanisms underlying E2F1 deregulation and found that the E2F sites in its promoter fail to repress its transcriptional activity in BL cells and that the transcriptional repressor E2F4 barely interacts with these sites. We also have found that E2F4 protein levels, but not those of its mRNA, are reduced in sBL cell lines relative to immortal B-cell lines. E2F4 protein expression is also decreased in 24 of 26 sBL tumor samples from patients compared with control tissues. Our data demonstrate that enforced E2F4 expression in BL cells not only diminishes E2F1 levels, but also reduces selectively the tumorigenic properties and proliferation of BL cells, while increasing their accumulation in G(2)/M. Our results therefore point to E2F4 as a target for developing novel and less toxic treatments for sBL.

Yin Yang 1 extends the Myc-related transcription factors network in embryonic stem cells.

The Yin Yang 1 (YY1) transcription factor is a master regulator of development, essential for early embryogenesis and adult tissues formation. YY1 is the mammalian orthologue of Pleiohomeotic, one of the transcription factors that binds Polycomb DNA response elements in Drosophila melanogaster and mediates Polycomb group proteins (PcG) recruitment to DNA. Despite several publications pointing at YY1 having a similar role in mammalians, others showed features of YY1 that are not compatible with PcG functions. Here, we show that, in mouse Embryonic Stem (ES) cells, YY1 has genome-wide PcG-independent activities while it is still stably associated with the INO80 chromatin-remodeling complex, as well as with novel RNA helicase activities. YY1 binds chromatin in close proximity of the transcription start site of highly expressed genes. Loss of YY1 functions preferentially led to a down-regulation of target genes expression, as well as to an up-regulation of several small non-coding RNAs, suggesting a role for YY1 in regulating small RNA biogenesis. Finally, we found that YY1 is a novel player of Myc-related transcription factors and that its coordinated binding at promoters potentiates gene expression, proposing YY1 as an active component of the Myc transcription network that links ES to cancer cells.

E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin.

Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll that exhibits cancer chemopreventive properties, but which also has been studied for its possible cancer therapeutic effects. We report here that human colon cancer cells treated with CHL accumulate in S-phase of the cell cycle, and this is associated with reduced expression levels of p53, p21, and other G(1)/S checkpoint controls. At the same time, E2F1 and E2F4 transcription factors become elevated and exhibit increased DNA binding activity. In CHL-treated colon cancer cells, bromodeoxyuridine pulse-chase experiments provided evidence for the inhibition of DNA synthesis. Ribonucleotide reductase (RR), a pivotal enzyme for DNA synthesis and repair, was reduced at the mRNA and protein level after CHL treatment, and the enzymatic activity was inhibited in a concentration-dependent manner both in vitro and in vivo. Immunoblotting revealed that expression levels of RR subunits R1, R2, and p53R2 were reduced by CHL treatment in HCT116 (p53(+/+)) and HCT116 (p53(-/-)) cells, supporting a p53-independent mechanism. Prior studies have shown that reduced levels of RR small subunits can increase the sensitivity of colon cancer cells to clinically used DNA-damaging agents and RR inhibitors. We conclude that by inhibiting R1, R2, and p53R2, CHL has the potential to be effective in the clinical setting, when used alone or in combination with currently available cancer therapeutic agents.

Differential expression of microRNA species in human gastric cancer versus non-tumorous tissues.

BACKGROUND AND AIM: MicroRNAs (miRNAs) play important roles in carcinogenesis. The global miRNA expression profile of gastric cancer has not been reported. The purpose of the present study was to determine the miRNA expression profile of gastric cancer. METHODS: Total RNA were first extracted from primary gastric cancer tissues and adjacent non-tumorous tissues and then small isolated RNAs (< 300 nt) were 3'-extended with a poly(A) tail. Hybridization was carried out on a microParaflo microfluidic chip (LC Sciences, Houston, TX, USA). After hybridization detection by fluorescence labeling using tag-specific Cy3 and Cy5 dyes, hybridization images were collected using a laser scanner and digitized using Array-Pro image analysis software (Media Cybernetics, Silver Spring, MD, USA). To validate the results and investigate the biological meaning of differential expressed miRNAs, immunohistochemistry was used to detect the differential expression of target genes. RESULTS: The most highly expressed miRNAs in non-tumorous tissues were miR-768-3p, miR-139-5p, miR-378, miR-31, miR-195, miR-497 and miR-133b. Three of them, miR-139-5p, miR-497 and miR-768-3p, were first found in non-tumorous tissues. The most highly expressed miRNAs in gastric cancer tissues were miR-20b, miR-20a, miR-17, miR-106a, miR-18a, miR-21, miR-106b, miR-18b, miR-421, miR-340*, miR-19a and miR-658. Among them, miR-340*, miR-421 and miR-658 were first found highly expressed in cancer cells. The expression of some target genes (such as Rb and PTEN) in cancer tissues was found to be decreased. CONCLUSION: To our knowledge, this is the first report about these miRNAs associated with gastric cancer. This new information may suggest the potential roles of these miRNAs in the diagnosis of gastric cancer.

Loss of 9p leads to p16INK4A down-regulation and enables RB/E2F1-dependent cell cycle promotion in gastrointestinal stromal tumours (GISTs).

Loss of chromosome 9p is a reliable predictor of malignant behaviour in gastrointestinal stromal tumours (GISTs). p16INK4A located at 9p21 inhibits the CDK4/6/cyclin D complex from phosphorylating RB. Phosphorylation of RB through CDK4/6/cyclin D in early G(1) phase frees the transcription factor E2F1 from RB and enables mRNA transcription of genes essential for G(1)/S phase transition. This study aims to determine the impact of 9p loss on mRNA and protein expression of p16INK4A and further key cell cycle regulators in the different phases of the cell cycle. Sixty primary GISTs previously characterized for 9p loss by comparative genomic hybridization were analysed for mRNA expression of p16INK4A, p15INK4B, CDK4, CDK6, cyclin D, p21CIP1p27KIP1, CDK2, cyclin E, cyclin B, RB and E2F1, using quantitative RT-PCR. The protein expression of CDK6, CDK2, p21CIP1, p27KIP1 and phosphorylated RB (S807/S811) was evaluated using protein arrays as a novel and highly sensitive platform for profiling of protein abundance and protein phosphorylation. In parallel, the nuclear percentages of immunohistochemical staining for p16INK4A, cyclin D, E2F1 and RB were quantified on a tissue microarray. GISTs with 9p loss had significantly higher proliferation rates, higher metastatic behaviour and shorter disease-free survival. On the molecular level, GISTs with 9p loss had a significantly reduced mRNA as well as nuclear protein expression of p16INK4A. RB was significantly more phosphorylated in these tumours, together with increased mRNA expression and nuclear staining for E2F1. Furthermore, GISTs with 9p loss had up-regulation of the late G1/S phase promoters CDK2 and cyclin E. We conclude that loss of 9p accompanied by early G1 phase inhibitor p16(INK4A) down-regulation in GISTs facilitates phosphorylation of RB, enabling E2F1-dependent transcription of genes essential for late G1/S phase transition. This study provides a possible basis for the accelerated proliferation and particularly malignant behaviour in GISTs with 9p loss.

Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer.

ZBTB7A (Pokemon) is a member of the POK family of transcriptional repressors. Its main function is the suppression of the p14ARF tumour suppressor gene. Although ZBTB7A expression has been found to be increased in various types of lymphoma, there are no reports dealing with its expression in solid tumours. Given that p14(ARF) inhibits MDM2, the main negative regulator of p53, we hypothesized that overexpression of ZBTB7A could lead indirectly to p53 inactivation. To this end, we examined the status of ZBTB7A and its relationship with tumour kinetics (proliferation and apoptosis) and nodal members of the p53 network in a panel of 83 non-small cell lung carcinomas (NSCLCs). We observed, in the majority of the samples, prominent expression of ZBTB7A in the cancerous areas compared to negligible presence in the adjacent normal tissue elements. Gene amplification (two- to five-fold) was found in 27.7% of the cases, denoting its significance as a mechanism driving ZBTB7A overproduction in NSCLCs. In the remaining non-amplified group of carcinomas, analysis of the mRNA and protein expression patterns suggested that deregulation at the transcriptional and post-translational level accounts for ZBTB7A overexpression. Proliferation was associated with ZBTB7A expression (p = 0.033) but not apoptosis. The association with proliferation was reflected in the positive correlation between ZBTB7A expression and tumour size (p = 0.018). The overexpression of ZBTB7A in both p53 mutant and p53 wild-type cases, implies either a synergistic effect or that ZBTB7A exerts its oncogenic properties independently of the p14(ARF)-MDM2-p53 axis. The concomitant expression of ZBTB7A with p14(ARF) (p = 0.039), instead of the anticipated inverse relation, supports the latter notion. In conclusion, regardless of the pathway followed, the distinct expression of ZBTB7A in cancerous areas and the association with proliferation and tumour size pinpoints a role for this novel cell cycle regulator in the pathogenesis of lung cancer.

[Comparative study of heterogeneity of extranodal and nodal diffuse large B cell lymphoma].

OBJECTIVE: Primary nodal and extranodal diffuse large B-cell lymphoma (DLBCL) were investigated for the heterogeneity of histopathology and immunophenotype, and their relation to clinical stage, comparatively. Whether E2F1 can be used as a germinal center B cell (GCB) DLBCL marker was also discussed. METHODS: Classification of histopathology and immunophenotype of 98 cases were studied by immunohistochemistry in tissue microarray. RESULTS: Histopathologic morphology presented as: centroblastic (CB,88.8%, 87/98), immunoblastic (IB,5.1%, 5/98), anaplastic (ALCL,3.1%, 3/98), and T cell rich B cell lymphoma (TCRBCL,3.1%, 3/98). Of which, 31 cases were GCB DLBCL, 10 (20.8%, 10/48) nodal, and 21 extranodal (42%, 21/50, P=0.024). The rates of Stages I/II in nodal and extranodal area were 48.5% and 70%, respectively (P=0.015). The rate of Stage I/ II in GCB DLBCL (74.2%) were higher than in non-GCB DLBCL (50.7%, P=0.029). The CD10 positive rates were 36.8% and 17.1% in Stages I/II and III/IV, respectively, and had significant differences (P=0.033). The CD10 positive rates were 18.8% and 38% in nodal and extranodal area, respectively (P=0.035). The positive rates of E2F1 were 38.8% and 16.5% in GCB and non-GCB DLBCL, respectively, and had significant differences (P=0.016). The positive rate of E2F1 had positive relation with the expression of CD10 and Bcl-6 (P<0.05). CONCLUSION: CB is the most type in 98 cases of DLBCL. The rate of GCB DLBCL was significant higher in extranodal than in nodal areas CD 10 can be used as a prognostic marker. The prognosis of GCB DLBCL is better than that of non-GCB DLBCL. The positive expression of E2F1 can be used as a marker of GCB DLBCL.

Activation of p38- and CRM1-dependent nuclear export promotes E2F1 degradation during keratinocyte differentiation.

E2F factors modulate a plethora of cell functions, including proliferation, differentiation, DNA repair and apoptosis. We have shown that differentiation in primary epidermal keratinocytes leads to E2F1 downregulation via activation of protein kinase C and p38 mitogen-activated protein kinase. We now demonstrate that E2F1 downregulation in differentiating keratinocytes involves its ubiquitination, as well as proteasomal degradation subsequent to CRM1-dependent nuclear export. E2F1 nuclear export specifically in response to differentiation requires regions adjacent to the cyclin A-binding domain in the N-terminus of this protein. Significantly, inhibition of p38 interferes with nuclear export and degradation of E2F1 during differentiation, but has no effect on E2F1 in undifferentiated cells. Thus, induction of differentiation in epidermal keratinocytes activates a specific program for post-transcriptional downregulation of E2F1, which involves signaling through p38 and activation of nuclear export pathways.

Proteomic analysis of the E2F1 response in p53-negative cancer cells: new aspects in the regulation of cell survival and death.

E2F1 is an essential transcription factor that regulates cell-cycle progression and apoptosis. Overexpression of E2F1 sensitizes neoplastic cells to apoptosis and leads to tumor growth suppression, making it an interesting target for anticancer therapy. Use of E2F1 as a therapeutic, however, requires a detailed knowledge of the mechanisms by which it controls cellular proliferation and apoptosis, and of other potential E2F1 activities. In this study, a differential proteome analysis was performed to identify proteins associated with E2F1 activity in inducible p53-deficient Saos-2ERE2F1 osteosarcoma cells. 2-DE revealed a distinct protein profile at 32 h after E2F1 activation. Thirty-three proteins were reproducibly identified as either up-regulated or down-regulated. Proteins were identified by MALDI-MS. They included hitherto unknown E2F1 target proteins of cytoskeletal origin, chaperones, enzymes, proteasomal proteins, and several heterogeneous nuclear ribonucleoproteins, suggesting its role in the ER-stress response, protein degradation, and modulation of pre-mRNA splicing. Protein analysis-derived results were verified by Western blot using representative protein candidates. Thirteen identified proteins were the products of genes known to be cancer related. Thus, proteome analysis provides new information about the complexity of E2F1 activities in human cancer cells that may be considered when using E2F1 as a drug.

Immunohistochemical detection of retinoblastoma protein and E2 promoter-binding factor-1 in ameloblastomas.

BACKGROUND: To clarify the roles of cell cycle regulation in oncogenesis and cytodifferentiation of odontogenic tumors, expression of retinoblastoma protein (RB) and E2 promoter-binding factor-1 (E2F-1) was analyzed in ameloblastomas as well as in tooth germs. METHODS: Tissue specimens of 10 tooth germs, 40 benign ameloblastomas, and five malignant ameloblastomas were examined immunohistochemically with the use of antibodies against RB, E2F-1, and phosphorylated RB. Ki-67 antigen immunostaining was made as a marker of cell proliferation. RESULTS: Immunohistochemical reactivity for RB, E2F-1, phosphorylated RB, and Ki-67 was detected in the nuclei of odontogenic epithelial cells near the basement membrane in tooth germs and benign and malignant ameloblastomas. The number of cells positive for phosphorylated RB was nearly equal to or slightly less than the number of cells positive for RB or E2F-1. The number of Ki-67-positive cells was slightly more than the numbers of cell positive for RB, E2F-1, or phosphorylated RB. The levels of immunoreactivity for RB, E2F-1, phosphorylated RB, and Ki-67 were slightly higher in benign and malignant ameloblastomas than in tooth germs. Plexiform ameloblastomas showed significantly higher expression of RB than follicular ameloblastomas. Ki-67 immunoreactivity was significantly higher in ameloblastic carcinomas than in metastasizing ameloblastomas. CONCLUSION: Similar immunoreactivity for RB, E2F-1, phosphorylated RB, and Ki-67 in tooth germs and ameloblastomas indicated cellular expression of phosphorylated RB and active-free E2F-1 in both normal and neoplastic odontogenic tissues. Expression of RB, E2F-1, and phosphorylated RB was considered to be involved in cell proliferation and differentiation of odontogenic epithelium via control of the cell cycle.

Transcription factor-mediated proliferation and apoptosis in benign and malignant thyroid lesions.

Transcription factors play an essential role in regulating both cell proliferation and programmed cell death. Proliferation and apoptosis-related transcription factor immunoexpression patterns were concomitantly investigated in tissue sections of normal thyroid, goiters, follicular adenomas and well-differentiated papillary and follicular carcinomas using antibodies against prothymosin alpha, E2F-1, p53, Bcl2, and Bax proteins. Proliferation and apoptotic indices were determined by Ki-67 immunoreactivity and the terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling technique, respectively. Prothymosin alpha and E2F-1 immunoexpression levels were found to be significantly elevated in well-differentiated carcinomas compared to adenomas, goiters and normal tissues (P < 0.05). Both proteins were directly correlated with the proliferation index (P < 0.05). E2F-1 was additionally correlated with the apoptotic index (P < 0.05). The majority of cases were negative for p53 staining. Positive Bcl2 immunostaining was detected in all thyroid histotypes. None of the normal tissues showed Bax immunoreactivity, while positive accumulation differed significantly between hyperplastic and neoplastic histotypes. Direct correlations were observed between prothymosin alpha and Bcl2 as well as between E2F-1 and Bax immunoexpression (P < 0.05). These data demonstrate that prothymosin alpha and E2F-1 are strongly involved in the proliferation processes of thyroid neoplasias. Furthermore, prothymosin alpha may promote cell survival through the Bcl2 anti-apoptotic pathway, while E2F-1-induced apoptosis via p53-independent pathways may be associated with transcriptional activation of bax pro-apoptotic gene.