Association Between TP53 Mutation and Prognosis in Wilms Tumor: A Meta-Analysis.

BackgroundTP53 mutation has been suggested to have prognostic value for patients with Wilms tumor (WT), but the results are still controversial. Methods: Relevant studies published until August 1, 2019 were identified by searching PubMed, EMBASE and Cochrane Library. A random-effect model was performed to assess pooled data. Begg's and Egger's test were used to evaluate the potential publication bias. Sensitivity analysis was used to evaluate the stability of results. Results: A total of seven eligible articles were included. There was no significant difference in the risk of death among patients with WT with different TP53 mutation status (odds ratio [OR] = 3.09, 95% confidence interval[CI]: 0.81-11.84). Combined hazard ratio (HR) suggested that TP53 mutation had an unfavorable impact on overall survival (OS) (HR = 4.17, 95% CI: 1.97-6.36) and disease-free survival (DFS) (HR = 2.23, 95% CI: 1.29-3.17) in WT. Conclusions: This meta-analysis demonstrates that TP53 mutations are associated with poorer prognosis in WT.

Celastrol inhibits colorectal cancer cell proliferation and migration through suppression of MMP3 and MMP7 by the PI3K/AKT signaling pathway.

Colorectal cancer (CRC) is one of the most frequent malignant tumors. Signaling by the PI3K/AKT pathway is crucial for CRC development and progression, including proliferation and migration. Celastrol has an anticancer effect, but its mechanism needs to be determined. Here, we showed that celastrol suppressed CRC cell proliferation and migration. Celastrol treatment also decreased the PI3K/AKT pathway components, and MMP3 and MMP7 expression levels. In addition, knockdown of AKT, not mTOR, inhibited MMP3 and MMP7 expression levels and AKT silencing promoted the celastrol-induced effects on CRC cell proliferation and migration. Taken together, these findings indicated that the celastrol-induced antitumor effects were mediated through MMP3 and MMP7 by the PI3K/AKT signaling pathway.

Effects of azimuthal angles on laser interference lithography.

This paper discusses the effects of azimuthal angles on two-, three-, and four-beam laser interference. In two- or three-beam laser interference, periodic surface structures of lines or dots were obtained. In four-beam laser interference with the polarization mode of TE-TM-TE-TM, the modulation in a particular direction was formed and calculated. In the work, a He-Ne laser system was used to simulate two-, three-, and four-beam laser interference, and the interference pattern was detected by a CCD. A high-power Nd:YAG laser interference lithography system was set up to pattern silicon wafers. In the experiments, one azimuthal angle was changed every time to form interference patterns when polarization states were fixed and incident angles were equal. The experimental results have shown that the azimuthal angle affects the periods and feature sizes of the interference patterns and the fabricated surface structures, which are in accordance with the theoretical and computer simulation results.

Fano resonance of nanoparticles embedded in Fabry-Perot cavities.

We present an optical structure, which consists of metal nanoparticles embedded in Fabry-Perot (F-P) cavity, to investigate the Fano resonance, which originates from the interaction between F-P mode and the plasmon modes supported by the nanoparticles. The coupling system is modeled theoretically by coupled-mode theory in time domain and the transmission properties are demonstrated numerically by the finite-difference time-domain method. The charge distribution features of the nanoparticle plasmon modes are further characterized by using boundary integral equation technology. Results show that the F-P modes can be used to active the optical inactive surface plasmon modes by breaking the mode symmetry.

Quantum dot-based immunochromatography test strip for rapid detection of Campylobacter jejuni.

Campylobacter jejuni is a recognized human foodborne pathogen which is one of the leading causes of human gastrointestinal enteritis worldwide. In stress conditions, C. jejuni is able to enter into a viable but non-culturable state that is difficult to diagnose. Hence a rapid, sensitive, and specific method is required to monitor food and water for natural or intentional contamination by this pathogen. We report a quantum dot (QD)-based immunochromatography test strip (QDITS) for rapid detection of C. jejuni. The QDITS is based on a sandwich type immunoassay with QD fluorescence for sensitive detection. Under optimized conditions, this QD-based fluorescence biosensor exhibits detection of 10(4) CFU/ml in pure culture of C. jejuni which is 10 times more sensitive than colloidal-gold ITS. The improved sensitivity is attributed to the high quantum yield and brightness of the photostable QDs. When the QDITS were tested in 206 chicken samples and 120 beef samples, this exhibited a specificity of 98.5% and 99.1% respectively. On these real samples, the sensitivity of the QDITS was 100% in agreement with traditional culture method. The QDITS that took only 10 minutes to complete hold promise for rapid, sensitive detection of C. jejuni in real samples without the need for sample preparations steps.

Speciation of organic phosphorus in a sediment profile of Lake Taihu. II. Molecular species and their depth attenuation.

The understanding of organic phosphorus (P) dynamics in sediments requires information on their species at the molecular level, but such information in sediment profiles is scarce. A sediment profile was selected from a large eutrophic lake, Lake Taihu (China), and organic P species in the sediments were detected using solution phosphorus-31 nuclear magnetic resonance spectroscopy (31P NMR) following extraction of the sediments with a mixture of 0.25 mol/L NaOH and 50 mmol/L EDTA (NaOH-EDTA) solution. The results showed that P in the NaOH-EDTA extracts was mainly composed of orthophosphate, orthophosphate monoesters, phospholipids, DNA, and pyrophosphate. Concentrations of the major organic P compound groups and pyrophosphate showed a decreasing trend with the increase of depth. Their half-life times varied from 3 to 27 years, following the order of orthophosphate monoesters > phospholipids > or = DNA > pyrophosphate. Principal component analysis revealed that the detected organic P species had binding phases similar to those of humic acid-associated organic P (NaOH-NRP(HA)), a labile organic P pool that tends to transform to recalcitrant organic P pools as the early diagenetic processes proceed. This demonstrated that the depth attenuation of the organic P species could be partly attributed to their increasing immobilization by the sediment solids, while their degradation rates should be significantly lower than what were suggested in previous studies.

A Nomogram and Risk Classification System Predicting the Prognosis of Patients with De Novo Metastatic Breast Cancer Undergoing Immediate Breast Reconstruction: A Surveillance, Epidemiology, and End Results Population-Based Study.

Background The lifespan of patients diagnosed with de novo metastatic breast cancer (dnMBC) has been prolonged. Nonetheless, there remains substantial debate regarding immediate breast reconstruction (IBR) for this particular subgroup of patients. The aim of this study was to construct a nomogram predicting the breast cancer-specific survival (BCSS) of dnMBC patients who underwent IBR. Methods A total of 682 patients initially diagnosed with metastatic breast cancer (MBC) between 2010 and 2018 in the Surveillance, Epidemiology, and End Results (SEER) database were included in this study. All patients were randomly allocated into training and validation groups at a ratio of 7:3. Univariate Cox hazard regression, least absolute shrinkage and selection operator (LASSO), and best subset regression (BSR) were used for initial variable selection, followed by a backward stepwise multivariate Cox regression to identify prognostic factors and construct a nomogram. Following the validation of the nomogram with concordance indexes (C-index), receiver operating characteristic (ROC) curves, calibration curves, and decision curve analyses (DCAs), risk stratifications were established. Results Age, marital status, T stage, N stage, breast subtype, bone metastasis, brain metastasis, liver metastasis, lung metastasis, radiotherapy, and chemotherapy were independent prognostic factors for BCSS. The C-indexes were 0.707 [95% confidence interval (CI), 0.666-0.748] in the training group and 0.702 (95% CI, 0.639-0.765) in the validation group. In the training group, the AUCs for BCSS were 0.857 (95% CI, 0.770-0.943), 0.747 (95% CI, 0.689-0.804), and 0.700 (95% CI, 0.643-0.757) at 1 year, 3 years, and 5 years, respectively, while in the validation group, the AUCs were 0.840 (95% CI, 0.733-0.947), 0.763 (95% CI, 0.677-0.849), and 0.709 (95% CI, 0.623-0.795) for the same time points. The calibration curves for BCSS probability prediction demonstrated excellent consistency. The DCA curves exhibited strong discrimination power and yielded substantial net benefits. Conclusions The nomogram, constructed based on prognostic risk factors, has the ability to provide personalized predictions for BCSS in dnMBC patients undergoing IBR and serve as a valuable reference for clinical decision making.

The autophagy-senescence connection in chemotherapy: must tumor cells (self) eat before they sleep?

Exposure of MCF-7 breast tumor cells or HCT-116 colon carcinoma cells to clinically relevant concentrations of doxorubicin (Adriamycin; Farmitalia Research Laboratories, Milan, Italy) or camptothecin results in both autophagy and senescence. To determine whether autophagy is required for chemotherapy-induced senescence, reactive oxygen generation induced by Adriamycin was suppressed by N-acetyl cysteine and glutathione, and the induction of ataxia telangiectasia mutated, p53, and p21 was modulated pharmacologically and/or genetically. In all cases, autophagy and senescence were collaterally suppressed. The close association between autophagy and senescence indicated by these experiments reflects their collateral regulation via common signaling pathways. The potential relationship between autophagy and senescence was further examined through pharmacologic inhibition of autophagy with chloroquine and 3-methyl-adenine and genetic ablation of the autophagy-related genes ATG5 and ATG7. However, inhibition of autophagy by pharmacological and genetic approaches could not entirely abrogate the senescence response, which was only reduced and/or delayed. Taken together, our findings suggest that autophagy and senescence tend to occur in parallel, and furthermore that autophagy accelerates the development of the senescent phenotype. However, these responses are not inexorably linked or interdependent, as senescence can occur when autophagy is abrogated.

Coupled-resonator-induced transparency in photonic crystal waveguide resonator systems.

We present an optical coupling system, which consists of waveguide, cavity and waveguide resonator, to investigate coupled-resonator-induced transparency effect. The transmission properties are analyzed theoretically by using coupled-mode theory in time domain. We also numerically demonstrate the effect by simulating the propagation of electromagnetic waves in photonic crystals by finite-difference time-domain method.

Expression of P-450(c11beta) in adrenal aldosterone-producing adenomas and nodular hyperplasia tissues.

BACKGROUND: Our previous study suggests that decreased P-450(c17alpha) expression correlated with the overproduction of aldosterone in APA and nodular hyperplasia in patients with primary aldosteronism. This study was performed to further investigate if P-450(c11beta) contributes to the overproduction of aldosterone in APA and nodular hyperplasia tissues. METHODS: Total RNA and protein were extracted from 7 cases of APA tissue, 3 nodular hyperplasia tissues, 7 normal adrenal glands. P-450(c11beta) mRNA was examined by dot blot and confirmed by Northern blot analysis and by realtime PCR. Protein expression level of P-450(c11beta) was also investigated by immunohistochemical staining and confirmed by Western blot. RESULTS: The relative expression level of P-450(c11beta) mRNA to beta-actin in APA, nodular hyperplasia and the normal adrenal gland group are 47 +/- 22%, 55 +/- 13%, 64 +/- 16% respectively by dot blot and are 94 +/- 18%, 101 +/- 20%, 112 +/- 62% respectively by Northern blot. These results are further confirmed by realtime PCR. This result was also supported by the relative protein expression level of P-450(c11beta) to beta-actin which are 118 +/- 15%, 107 +/- 32%, 108 +/- 22% respectively evaluated by Western blot. There was no significant difference in protein expression level of P-450(c11beta) among the normal adrenal gland tissues, APA and adrenal nodular hyperplasia tissue, either (P > 0.05). CONCLUSIONS: These results suggest that P-450(c11beta) is not a key contributor to the overproduction of aldosterone in APA and nodular hyperplasia and can not be considered as a potential marker to differentiate between them in patients with primary aldosteronism.

Mst2 Overexpression Inhibits Thyroid Carcinoma Growth and Metastasis by Disrupting Mitochondrial Fitness and Endoplasmic Reticulum Homeostasis.

Although the incidence of thyroid carcinoma has increased over the past several decades, it has an excellent prognosis and overall 5-year survival, with a stable mortality rate, except in cases with advanced stages or rare malignant tumor types. Biomarkers have emerged as effective targets of molecular therapy against thyroid carcinoma due to their rapid and convenient detection; however, there has been little clinical application. Macrophage stimulating 2 (Mst2) is a proapoptotic protein with implications in carcinogenesis and metastasis. We found that Mst2 overexpression-induced endoplasmic reticulum (ER) stress in MDA-T32 thyroid carcinoma cells, accompanied by elevated caspase-12 activity, increased apoptotic rate, and reduced cell viability. In addition, Mst2 overexpression contributed to mitochondrial damage, as evidenced by increased mitochondrial oxidative stress and activated the mitochondrial apoptotic pathway. Inhibition of the JNK pathway abolished these effects. These results show Mst2 to be a novel tumor suppressor that induces mitochondrial dysfunction and ER stress via the JNK pathway. Thus, Mst2 could potentially serve as a biomarker for developing targeted therapy against thyroid carcinoma.

Influence of the phosphodiesterase-5 inhibitor, sildenafil, on sensitivity to chemotherapy in breast tumor cells.

Studies were performed to determine the influence of the phosphodiesterase-5 inhibitor, sildenafil, on sensitivity to adriamycin (doxorubicin) in four human breast tumor cell lines and one murine breast tumor line. Sildenafil did not interfere with the effectiveness of adriamycin in any of the cell lines tested. Sildenafil also failed to protect MDA-MB231 cells against the cytotoxicity of cisplatin, taxol or camptothecin. Sildenafil enhanced sensitivity to adriamycin markedly in the p53 mutant MDA-MB231 and p53 null MCF-7/E6 cells and moderately in the MCF-7/caspase 3 and 4T1 cell lines. In the MDA-MB231 cells, sildenafil increased the extent of DNA damage induced by adriamycin as well as the extent of apoptotic cell death. Sildenafil did not influence sensitivity to adriamycin in bone marrow cells or macrophages. In an immunocompetent model of breast cancer (4T1 mammary carcinoma in Balb/c mice), sildenafil did not attenuate the antitumor effects of adriamycin; furthermore, the combination of sildenafil with adriamycin was no more toxic to the animals than adriamycin alone. Given that sildenafil has been shown to have the potential to protect the heart against the toxicity of adriamycin, these studies suggest that the inclusion of sildenafil with conventional chemotherapeutic protocols involving adriamycin (and possibly cisplatin, camptothecin and/or paclitaxel) should not compromise the antitumor effectiveness of these drugs nor enhance their toxicity to the patient.

FDA Approval Summary: Calaspargase Pegol-mknl For Treatment of Acute Lymphoblastic Leukemia in Children and Young Adults.

On December 20, 2018, the Food and Drug Administration approved calaspargase pegol-mknl (CALASP), an asparagine-specific enzyme, as a component of a multi-agent chemotherapeutic regimen for acute lymphoblastic leukemia (ALL) in pediatric and young adult patients age 1 month to 21 years. Efficacy was determined on the basis of achievement and maintenance of steady-state nadir serum asparaginase activity (NSAA) above 0.1 U/mL when using CALASP, 2,500 U/m2 intravenously, every 3 weeks. In a randomized comparison to pegaspargase (PEGASP) every 2 weeks, treatment with CALASP every 3 weeks had a similar safety profile and no substantial impairment in event-free survival. The pharmacokinetics of CALASP were studied when administered in combination with multiagent chemotherapy in 124 patients with B-cell ALL in Study AALL07P4 and Study DFCI 11-001. The results showed that 123 [99%, 95% confidence interval (CI), 96%-100%] of the 124 patients maintained NSAA >0.1 U/mL at weeks 6, 12, 18, 24, and 30 of post-induction phase. Maintaining adequate NSAA levels is critical to successful treatment of ALL. Herein, we describe the FDA review and approval of CALASP.See related commentary by Lew, p. 325.

Downregulation of Long Noncoding RNA HOTAIR and EZH2 Induces Apoptosis and Inhibits Proliferation, Invasion, and Migration of Human Breast Cancer Cells.

BACKGROUND: The long noncoding RNA HOTAIR (HOX transcript antisense intergenic RNA) has been reported to be a biomarker for various malignant tumors; however, its involvement in breast cancer is not fully understood. The aim of this study was to investigate the effects involved with long noncoding RNA HOTAIR and EZH2 (enhancer of zeste homologue 2) on the processes of proliferation, invasion, migration, and apoptosis of breast cancer cells. MATERIALS AND METHODS: The expressions of HOTAIR and EZH2 in both normal human mammary epithelial cell (HBL-100) and breast cancer cell lines (MCF-7, MDA-MB-231, and SKBR-3) were detected by means of reverse transcription-quantitative polymerase chain reaction. The MCF-7 cells that exhibited the highest HOTAIR expressions were selected for further studies and divided into the control, negative control, and small interfering RNA-HOTAIR groups. The proliferation, invasion, migration, and apoptosis of breast cancer cells were evaluated by MTT assay, Scratch test, Transwell assay, and flow cytometry, respectively. The combination of HOTAIR with EZH2 and PTEN was predicted by bioinformation, with a dual-luciferase reporter gene assay providing further verification. RESULTS: Initially, lower expressions of HOTAIR and EZH2 in the normal human mammary epithelial cells, while higher expressions in the breast cancer cells of MCF-7, MDA-MB-231, and SKBR-3 were detected. In addition, the downregulation of HOTAIR or silencing of EZH2 was revealed to repress the proliferation, invasion, and migration, while acting to promote the apoptosis of the breast cancer cells. Furthermore, HOTAIR could bind specifically to EZH2 and PTEN, highlighting the capability of HOTAIR to inhibit the expression of PTEN by recruiting EZH2 in breast cancer, while the TCGA database demonstrated the expressions of PTEN were lower in breast cancer cells. CONCLUSIONS: The study suggests the higher expressions of HOTAIR and EZH2 among three breast cancer cells. Furthermore, the downregulation of HOTAIR or silencing of EZH2 was noted to inhibit the proliferation, invasion, and migration of breast cancer cells, while promoting their apoptosis.

Erythropoietin fails to interfere with the antiproliferative and cytotoxic effects of antitumor drugs.

PURPOSE: Erythropoietin (EPO) therapy is widely used for the prevention and treatment of anemia resulting from cancer chemotherapy. Native EPO regulates erythropoiesis, at least in part, by protecting erythroid progenitor cells from apoptotic cell death. The recent discovery of the EPO receptor (EPOR) on cancer cells raises the concern that EPO therapy might stimulate tumor growth and/or protect cancer cells from drug-induced apoptosis. Therefore, the capacity of EPO to interfere with the effects of conventional chemotherapeutic drugs on proliferation, apoptosis, and the induction of senescence was investigated in MCF-7 and MDA-MB231 breast tumor cells, which express the EPOR as well as in F-MEL erythroleukemia cells. EXPERIMENTAL DESIGN: Breast cancer cells and F-MEL leukemic cells were cultured in the presence or absence of EPO and then exposed to antitumor drugs. Cell proliferation was assessed by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay 72 hours after drug exposure. Cytotoxicity was monitored by clonogenic survival. Apoptosis was evaluated either by the terminal deoxyribonucleotide transferase-mediated nick-end labeling assay or fluorescence-activated cell sorting analysis, and senescence was monitored by beta-galactosidase staining. EPO signaling was assessed by monitoring the phosphorylation/activation of specific signaling proteins. RESULTS: EPO failed to stimulate the proliferation of MCF-7 or MDA-MB231 breast tumor cells or F-MEL leukemic cells. EPO treatment also failed to interfere with the antiproliferative and/or cytotoxic effects of Adriamycin, Taxol, and tamoxifen in breast tumor cells (or of cytarabine and daunorubicin in F-MEL cells). EPO failed to prevent apoptosis induced by Taxol or senescence induced by Adriamycin in MCF-7 cells. EPO stimulated the activation of extracellular signal-regulated kinase, p38, and c-Jun-NH(2)-kinase in MCF-7 cells but did not activate Akt or signal transducers and activators of transcription 5 (STAT5). EPO failed to activate any of these signaling pathways in MDA-MB231 cells. Cytarabine and daunorubicin interfered with EPO signaling in F-MEL cells. CONCLUSIONS: These findings suggest that EPO is unlikely to directly counteract the effectiveness of cancer chemotherapeutic drugs. This may be a consequence of either ineffective signaling through the EPOR or drug-mediated suppression of EPO signaling.

Potentiation of radiation sensitivity in breast tumor cells by the vitamin D3 analogue, EB 1089, through promotion of autophagy and interference with proliferative recovery.

1,25-Dihydroxyvitamin D(3) and vitamin D(3) analogues, such as EB 1089, potentiate the response to ionizing radiation in breast tumor cells. The current studies address the basis for this interaction by evaluating DNA damage and repair, the effect of interference with reactive oxygen generation, the involvement of p53 and caspase-3, signaling through c-myc, as well as the induction of senescence and multiple modes of cell death. EB 1089 failed to increase the extent of radiation-induced DNA damage or to attenuate the rate of DNA repair. The reactive oxygen scavengers N-acetyl-l-cysteine and reduced glutathione failed to protect the cells from the promotion of cell death by EB 1089 and radiation. Whereas MCF-7 cells expressing caspase-3 showed significant apoptosis with radiation alone as well as with EB 1089 followed by radiation, EB 1089 maintained its ability to confer susceptibility to radiation-induced cell killing, in large part by interference with proliferative recovery. In contrast, in breast tumor cells lacking p53, where radiation promoted extensive apoptosis and the cells failed to recover after radiation treatment, EB 1089 failed to influence the effect of radiation. EB 1089 treatment interfered with radiation-induced suppression of c-myc; however, induction of c-myc did not prevent senescence by radiation alone or radiation-induced cell death promoted by EB 1089. EB 1089 did not increase the extent of micronucleation, indicative of mitotic catastrophe, induced by radiation alone. However, EB 1089 did promote extensive autophagic cell death in the irradiated cells. Taken together, these studies suggest that the effect of EB 1089 treatment on the radiation response is related in part to enhanced promotion of autophagic cell death and in part to interference with the proliferative recovery that occurs with radiation alone in p53 wild-type breast tumor cells.

Evasion of a single-step, chemotherapy-induced senescence in breast cancer cells: implications for treatment response.

PURPOSE: The purpose of this study is to define the mechanistic basis for recovery of proliferative capacity in breast tumor cells after chemotherapy. Here, we test the hypothesis that evasion of senescence confers resistance to chemotherapeutic drugs and ionizing radiation. EXPERIMENTAL DESIGN: MCF-7 cells were treated with a single, clinically relevant dose (0.75-1.0 micromol/L) of Adriamycin. Two weeks following induction of senescence, clonal outgrowths were expanded and characterized in terms of senescence-associated beta-galactosidase activity, gene expression profiles (Affymetrix U95 probe sets, Affymetrix, Santa Clara, CA) with confirmatory Western analyses, and telomerase activity following a second drug treatment. Levels of intracellular Adriamycin, as well as cross-resistance to other therapeutic agents, were also determined to define the resistance phenotype. RESULTS: A senescence-resistant (SR) clone (clone 2) was identified that was largely refractory to both Adriamycin-induced and gamma-irradiation-induced senescence. Clone 2 continued to proliferate and maintain high levels of telomerase activity following a second drug treatment, when treated parental cells expressed very low levels of telomerase and many positive cell cycle regulators. SR clone 2 also expressed substantially more cdc-2 than parental cells and undetectable levels of MDR1, showed an intact p53 checkpoint and only a modestly lower level of intracellular drug accumulation, while exhibiting cross-resistance to other topoisomerase inhibitors. CONCLUSIONS: SR clone 2 is intrinsically resistant to DNA damage-induced senescence perhaps through an ability to prevent down-regulation of cdc-2. Telomerase is a marker of proliferative recovery for breast cancer cells after chemotherapy exposure. Evasion or escape from a single-step, drug-induced senescence may represent a unique and previously unrecognized drug-resistance phenotype.

Influence of p53 and caspase 3 activity on cell death and senescence in response to methotrexate in the breast tumor cell.

The influence of p53 function and caspase 3 activity on the capacity of the antifolate, methotrexate, to promote senescence arrest and apoptotic cell death was investigated in breast tumor cells. In p53 wild-type, but caspase 3 deficient MCF-7 breast tumor cells, death of approximately 40% of the cell population was observed immediately after acute exposure to 10 microM methotrexate (the IC80 value for a 2 h drug exposure). There was no evidence of either DNA fragmentation, a sub G0 population or morphological alterations indicative of apoptosis; however, PARP cleavage was detected. Cell death was succeeded by growth arrest for at least 72 h--where arrest was characterized by expression of the senescence marker, beta-galactosidase. The response to methotrexate in MCF-7/E6 cells with attenuated p53 function was also primarily growth arrest--but lacking characteristics of senescence. In contrast, MCF-7 cells which expressed caspase 3 demonstrated a gradual and continuous loss of cell viability and unequivocal morphological evidence of apoptosis. DNA fragmentation indicative of apoptosis was also detected after exposure to methotrexate in p53 mutant MDA-MB231 breast tumor cells which also express caspase 3. Methotrexate-induced both p53 and p21waf1/cip1 in MCF-7 cells within 6 h; however, no significant DNA strand breakage was evident before 18 h, suggesting that the induction of p53 reflects a response to cellular stress other than DNA damage, such as nucleotide depletion. Overall, these studies suggest that the nature of the cellular response to methotrexate depends, in large part, on p53 and caspase function. p53 appears to be required for methotrexate-induced senescence, but not apoptosis, caspase 3 is required for DNA fragmentation and the morphological changes associated with apoptosis, while neither p53 nor caspase 3 are required for methotrexate-induced growth arrest. Furthermore, the senescence phenotype may occur in the absence of direct DNA damage.

[The transcriptional regulation study on human delta globin gene with CAAT box C-->T point mutation in its promoter].

OBJECTIVE: To study the transcriptional regulation of human delta globin gene with C-->T point mutation at -64 in its promoter. METHODS: Human delta globin genes including wild CAAT box and mutant CAAT box (-64C-->T) were separately cloned into eukaryotic expression vector pcDNA3.1 (-)/Myc-His A which was cut out the strong promoter CMV, transfected MEL cells, and induced by DMSO to express. The transcriptional regulation of human delta globin gene was analysed using semi-quantitative RT-PCR. RESULTS: The expression level of human delta globin gene with mutant CAAT box was 2.2-fold as high as that with wild CAAT box. CONCLUSION: The defective CAAT box of human delta globin gene promoter region may be one of the major reasons for its low expression level.

[Gene therapy of rat prolactinomas mediated by adenoviral vectors with rat tyrosine hydroxylase gene].

OBJECTIVE: To investigate the potential of gene therapy of rat prolactinomas mediated by adenoviral vectors with a gene encoding rat tyrosine hydroxylase. METHODS: Recombinant replication-deficient adenovirus named Ad-GFP-TH with rat TH-cDNA and control adenovirus named Ad-GFP were constructed by homologous recombination in bacterial cells. The rat pituitary prolactinoma cell line MMQ are chosen as the target cells to study the effect of gene therapy on their growth and prolactin secretion mediated by Ad-GFP-TH. RESULTS: Recombinant Ad-GFP-TH and Ad-GFP were successfully reconstructed. Transfection of MMQ cells with Ad-GFP-TH not only restrained their growth but also decreased their PRL secretion. CONCLUSION: Gene therapy may serve for a potential treatment for prolactinomas, especially invasive prolactinomas.