[Clinicopathological Features of Primary Small Cell Neuroendocrine Carcinoma of the Bladder].

Objective To investigate the clinicopathological features,immunohistochemical features,diagnosis,and relationship with sporadic prostate cancer in primary small cell neuroendocrine carcinoma of the bladder. Methods We retrospectively analyzed the clinical characteristics of 12 patients with primary small cell neuroendocrine carcinoma of the bladder diagnosed at Beijing Chao-Yang Hospital affiliated to Capital Medical University from January 2013 to September 2022.The histological features of primary small cell neuroendocrine carcinoma of the bladder were re-evaluated by two pathologists according to the 2022 revision of the World Health Organization Classification of Tumors of the Urinary System and Male Genital Organs.Electronic medical records were retrieved,and telephone follow-up was conducted from the time of histopathological diagnosis to the death or the end of the last follow-up until January 31,2023. Results The 12 patients include 7 patients in pT3 stage and 1 patient in pT4 stage.Eight patients were complicated with other types of tumors,such as high-grade urothelial carcinoma of the bladder and squamous cell carcinoma.Five patients had sporadic prostate cancer.Immunohistochemical staining showed that 12 (100.0%),10 (83.3%),and 8 (66.7%) patients were tested positive for CD56,Syn,and CgA,respectively.The Ki67 proliferation index ranged from 80% to 90%.Five patients with urothelial carcinoma were tested positive for CK20,GATA3,and CK7.P504S was positive in all the 5 patients with prostate cancer,while P63 and 34ßE12 were negative.The follow-up of the 12 patients lasted for 3-60 months.Eight of these patients died during follow-up,with the median survival of 15.5 months.Four patients survived. Conclusions Primary small cell neuroendocrine carcinoma of the bladder is a rare urological tumor with high aggressiveness and poor prognosis.In male patients with bladder prostatectomy,all prostate tissue should be sampled.If prostate cancer is detected,the prostate-specific antigen level should be monitored.

Evaluation of Physiological Coping Strategies and Quality Substances in Purple SweetPotato under Different Salinity Levels.

Although salinity stress is one of the principal abiotic stresses affecting crop yield, a suitable concentration of NaCl has proven to be useful for increasing crop quality. This study used low salinity (34 mmol/L NaCl) and high salinity (85 mmol/L) to cultivate purple sweetpotato. Using transcriptomics and metabolomics to profile the pathway indicated that glycometabolism, secondary metabolite biosynthesis and the starch catabolic process were the significant pathways under the salinity stress. Further research showed that purple sweetpotato could regulate genes related to the regulation of the cellular Na+, K+, and other ions concentration in response to the low salinity tolerance, but loses this ability under high salinity. Meanwhile, under low salinity, the activity of antioxidant enzymes and their related gene expression are maintained at a high level. The low salinity influences the monosaccharide composition as well as the content and regulation of genes related to starch synthesis. Quality analysis showed that the low salinity could increase the starch content and influence the amylopectin biosynthesis. It suggested that low salinity promotes substance accumulation. High salinity could increase the anthocyanins biosynthesis and low salinity had a significant impact on phenolic acid and flavonol. Finally, the gene expression levels also prove the low salinity could change the composition and content level of the purple sweetpotato. This study showed that an appropriate concentration of NaCl can be used as an elicitor for application in purple sweetpotato planting.

Comparative transcriptome analysis reveals candidate genes involved in anthocyanin biosynthesis in sweetpotato (Ipomoea batatas L.).

Sweetpotato [Ipomoea batatas (L.) Lam] is an economically important crop for fresh and processed consumption and is widely cultivated worldwide, especially in China. Various sweetpotato cultivars with different storage root colors are presently available. The purple-fleshed sweetpotato obtains its color from anthocyanin accumulation in the storage roots, which is beneficial for both plant and human health. To date, the molecular mechanism of this anthocyanin accumulation has not been studied in detail. In our study, three cDNA libraries generated from 'Xuzi8' with dark-purple flesh, 'Xuzi6' with light-purple flesh, and 'Xu28' with white flesh were sequenced utilizing an Illumina HiSeq™ 2500 platform. Corresponding totals of 28,093,466, 29,239,729 and 27,217,440 raw reads were obtained from the three libraries and assembled into 137,625 unigenes with an average length of 481 bp. Moreover, 79,203 unigenes (57.55%) were found to be annotated in several public databases, and 1285 unigenes were differentially expressed among the Xu28 vs Xuzi8, Xu28 vs Xuzi6, and Xuzi6 vs Xuzi8 libraries. After functional category enrichment analysis of differential expression genes (DEGs), 25 genes were selected as the candidate genes related to anthocyanin accumulation. Furthermore, the expression patterns of some selected DEGs were verified by quantitative real-time PCR (qRT-PCR), and the correlation between expression levels of relevant genes involved in anthocyanin biosynthesis and anthocyanin content was determined. Taken together, the results compose a transcriptomic analysis to investigate the differences in purple flesh formation in the storage roots among different sweetpotato varieties, with the notable outcome that several key genes can now be closely linked to anthocyanin biosynthesis.

A novel glutathione S-transferase gene from sweetpotato, IbGSTF4, is involved in anthocyanin sequestration.

Anthocyanins are synthesized by multi-enzyme complexes localized at the cytoplasmic surface of the endoplasmic reticulum (synthesis site), and transported to the destination site, the vacuole. Three mechanisms for the vacuolar accumulation of anthocyanin in plant species have been proposed. Previous studies have indicated that glutathione S-transferase (GST) genes from model and ornamental plants are involved in anthocyanin transportation. In the present study, an anthocyanin-related GST, IbGSTF4, was identified and characterized based on transcriptome results. Phylogenetic analysis revealed that IbGSTF4 was most closely correlated to PhAN9 and CkmGST3, the anthocyanin-related GST of Petunia hybrida and Cyclamen. Furthermore, the expression analysis revealed that IbGSTF4 is strongly expressed in pigmented tissues, when compared to green organs, which is in agreement to the ability to correlate with anthocyanin accumulation. A GST activity assay uncovered that the IbGST4 protein owned similar activities with the GST family. Furthermore, the molecular functional complementation of Arabidopsis thaliana mutant tt19 demonstrated that IbGSTF4 might play a vital role in the vacuole sequestration of anthocyanin in sweetpotato. Moreover, the dual luciferase assay revealed that the LUC driven by the promoter of IbGSTF4 could not be directly activated by IbMYB1, suggesting that the regulatory mechanism of anthocyanin accumulation and sequestration in sweetpotato was intricate.

Clinical characteristics of autoimmune rheumatic disease-related organizing pneumonia.

To study the clinical characteristics of autoimmune rheumatic disease-related organizing pneumonia (AIRD-OP), the clinical presentation, radiological findings, treatment, and outcome of AIRD-OP patients were analyzed, in comparison with patients with cryptogenic organizing pneumonia (COP). A total of 131 OP patients were identified, including 57 cases of AIRD-OP, 35 cases of COP, and 39 cases of other disease-related OPs. Among AIRD-OP patients, 36 (63%) presented the symptoms of OP at onset. The primary disease of AIRDs included Sjogren's syndrome (38%), polymyositis/dermatomyositis (23%), rheumatoid arthritis (23%), and undifferentiated AIRD. Compared with COP patients, the prevalence of patients having cough and malaise at baseline was significantly lower (54.4 vs 82.9%, P < 0.05; 49.1 vs 70.6%, P < 0.05), and the signs of moist rales and crackles were more common in AIRD-OP patients (54.4 vs 32.4%, P < 0.05; 49.1 vs 26.5%, P < 0.05). Lung function (TLC%, FVC%) was more significantly reduced in AIRD-OP patients (72 vs 97%, P < 0.05;75 vs 96%, P < 0.05). The dosage of corticosteroids prescribed was significantly higher in AIRD-OP patients (44 vs 37 mg/day, P < 0.05). The complete recovery rate was slightly lower in AIRD-OP patients (22.2 vs 29%, P > 0.05) with a tendency towards higher recurrence rate in AIRD-OP patients (32.7 vs 14.3%, P < 0.05). AIRD-OP may be the most common cause of OP. OP can be the initial presentation of AIRD. Compared with COP patients, AIRD-OP patients are characterized with occult onset but more severe lung involvement and higher recurrence rate.

Cloning and characterization of a novel GIGANTEA gene in sweet potato.

The transition from vegetative to reproductive growth, a key event in the lifecycle of a plant, is affected by environmental stresses. The flowering-time regulator GIGANTEA (GI) may be contributing to susceptibility of the regulation of photoperiodic flowering, circadian rhythm control, and abiotic stress resistance in Arabidopsis. However, the role of GI in sweet potato remains unknown. Here, we isolated and characterized a GI gene (IbGI) from sweet potato (Ipomoea batatas [L.] Lam). The IbGI cDNA sequence was isolated based on information from a sweet potato transcriptome database. IbGI mRNA transcript levels showed robust circadian rhythm control during the light-dark transition, and the expression of IbGI was stronger in leaves and roots than in stems. IbGI protein is predominantly localized to the nucleus. IbGI expression was upregulated by high temperature, drought, and salt stress but downregulated by cold stress. Overexpressing IbGI in the Arabidopsis gi-2 mutant background rescued its late flowering phenotype and reduced its salt tolerance. Taken together, these results indicate that IbGI shares functions in regulating flowering, the circadian rhythm, and tolerance to some stresses with other GI orthologs.

Overexpression of CuZnSOD and APX enhance salt stress tolerance in sweet potato.

Abiotic stresses cause accumulation of reactive oxygen species (ROS) in plants, CuZnSOD and APX are first line defenses against ROS caused by oxidative stress. In this study, CuZnSOD and APX were transferred into salt sensitive sweet potato (cv. Xushu 55-2) under control of stress inducible SWPA2 promoter and tolerance to salt stress was evaluated. When 100 mM NaCl was used to treat stem cuttings, transgenic plants showed enhanced tolerance compared to wild type (WT) plants. Rooting was significantly retarded in WT plants whereas all transgenic plants had significantly enhanced root growth under salt stress. Integration of SOD gene was confirmed by southern blot analysis, and the copy number ranged from 1 to 3. The expression levels of CuZnSOD and APX in transgenic plants were significantly increased up to 13.3 and 7.8 folds to WT under salinity conditions, respectively. SOD and APX activity and ROS staining showed enzyme activities of transgenic plants were increased under salt stress. These results show that CuZnSOD and APX have important roles in enhancing the salt tolerance of sweet potato.

Effect of Embelin on TRAIL receptor 2 mAb-induced apoptosis of TRAIL-resistant A549 non-small cell lung cancer cells.

INTRODUCTION: Some non-small cell lung cancer (NSCLC) tumor cells are insensitive to tumor necrosis factor- related apoptosis-inducing ligand (TRAIL) -based therapy. This study was conducted to examine the effect of embelin on the sensitivity of the A549 NSCLC cell line to TRAIL receptor2 (TRAILR2) monoclonal antibodies and to investigate the potential mechanisms. MATERIALS AND METHODS: A549 cells were treated with embelin, TRAILR2 mAb or a combination of both. Cell viability was measured using ATPlite assay and apoptosis rates were determined by flow cytometry with AnnexinV-FITC and propidium iodide staining, with the expression levels of proteins analyzed by Western blotting. RESULTS: The cell survival rate of separate treatments with 100 ng/ml TRAILR2 antibody or 25 uM embelin were 81.5±1.57% and 61.7±2.84%, respectively. Their combined use markedly decreased cell viability in A549 cells to 28.1±1.97% (P<0.05). The general caspase inhibitor Z-VAD- FMK could inhibit the embelin-enhanced sensitivity of A549 cells to TRAILR2 mAb (75.97±3.17%)(P<0.05). Both flow cytometry and cell morphological analysis showed that embelin was able to increase TRAIL-induced apoptosis in A549 cells. Combined treatment with embelin and TRAILR2 mAb augmented the activation of initiator caspases and effector caspase. In addition, A549 cells showed increasing levels of TRAILR2 protein and decreasing levels of Bcl-2, survivin and c-FLIP following the treatment with embelin+TRAILR2 mAb. CONCLUSIONS: Embelin could enhance TRAIL-induced apoptosis in A549 cells. The synergistic effect of the combination treatment might be due to modulation of multiple components in the TRAIL receptor-mediated apoptotic signaling pathway, including TRAILR2, XIAP, survivin, Bcl-2 and c-FLIP.

Evaluation of ALK rearrangement in Chinese non-small cell lung cancer using FISH, immunohistochemistry, and real-time quantitative RT- PCR on paraffin-embedded tissues.

Patients with ALK gene rearrangements often manifest dramatic responses to crizotinib, an ALK inhibitor. Accurate identification of patients with ALK-positive non-small cell lung cancer (NSCLC) is essential for the clinical application of ALK-targeted therapy. However, assessing EML4-ALK rearrangement in NSCLC remains challenging in routine pathology practice. The aim of this study was to compare the diagnostic accuracy of FISH, immunohistochemistry (IHC), and real-time quantitative RT-PCR (QPCR) methodologies for detection of EML4-ALK rearrangement in NSCLC and to appraise immunohistochemistry as a pre-screening tool. In this study, a total of 473 paraffin-embedded NSCLC samples from surgical resections and biopsies were analyzed by IHC with ALK antibody. ALK rearrangement was further confirmed by FISH and QPCR. ALK protein expression was detected in twenty patients (20/473, 4.2%). Of the 20 ALK-positive cases by IHC, 15 cases were further confirmed as ALK rearrangement by FISH, and 5 cases were not interpretable. Also, we evaluated 13 out of the 20 IHC-positive tissues by QPCR in additional to FISH, and found that 9 cases were positive and 2 cases were equivocal, whereas 2 cases were negative although they were positive by both IHC and FISH. The ALK status was concordant in 5 out of 8 cases that were interpretable by three methods. Additionally, none of the 110 IHC-negative cases with adenocarcinoma histology showed ALK rearrangements by FISH. Histologically, almost all the ALK-rearranged cases were adenocarcinoma, except that one case was sarcomatoid carcinoma. A solid signet-ring cell pattern or mucinous cribriform pattern was presented at least focally in all ALK-positive tumors. In conclusion, our findings suggested that ALK rearrangement was associated with ALK protein expression. The conventional IHC assay is a valuable tool for the pre-screening of patients with ALK rearrangement in clinical practice and a combination of FISH and QPCR is required for further confirmation.

[Pulmonary capillary hemangiomatosis: a clinicopathologic analysis of 2 cases with review of literature].

OBJECTIVE: To investigate the clinicopathologic features of pulmonary capillary hemangiomatosis (PCH). METHODS: The clinical and pathologic profiles of 2 PCH cases were evaluated. Immunohistochemical study (EnVision method) was performed on fixed tissues. The biologic behavior was analyzed with follow-up data. RESULTS: The main presenting symptom was dyspnea. Chest radiography of the two cases depicted diffuse, ground-glass nodules, accompanied by enlarged central pulmonary arteries. Microscopically, the most distinctive feature was proliferation of capillary channels within pulmonary interstitium and alveolar walls, accompanied by muscularization of arterioles. Immunohistochemical study showed an abundance of mast cells in the lesion, and staining for platelet-derived growth factor receptor-beta (PDGFR-ß) localized to vascular smooth muscles surrounding the proliferating capillaries and the mast cells. The index of Ki-67 was less than 1 percent and the p53 was negative. CONCLUSIONS: PCH is a rare vascular proliferative disease of yang patients. Increased number of mast cell and the up-regulation of PDGFR-ß may suggest mechanism for PCH. The clinical and radiologic diagnosis of PCH can be very difficult, and the histological examination is regarded as the most reliable means to establish the diagnosis. Pathologists should improve their knowledge on PCH.

[Pathologic diagnosis and clinical analysis of chronic extrinsic allergic alveolitis].

OBJECTIVE: To study the clinicopathologic features and diagnostic approach of chronic extrinsic allergic alveolitis (EAA). METHODS: Seven cases of chronic EAA diagnosed by open lung biopsy or lung transplant were enrolled into the study. The clinical and pathologic features were analyzed and the literature was reviewed. RESULTS: There were altogether 4 men and 3 women. The age of the patients ranged from 30 to 65 years (mean = 48 years). All cases represented chronic form and five cases diagnosed by open lung biopsy also showed features of recent aggravation, leading to hospitalization. Four cases had known history of exposure to inciting gases, pollens and pets, and only 2 cases were positive for allergens. High-resolution CT scan showed ground-glass attenuation and reticular pattern that often had a patchy distribution and central predominance. Bronchoalveolar lavage analysis showed marked lymphocytosis, with CD4(+)/CD8(+) ratio less than 1. Lung function test demonstrated a restrictive ventilatory defect, with decreased compliance, reduced diffusion capacity and high airway obstruction. Five cases had open lung biopsy performed and two cases had undergone lung transplantation. Pathologic examination showed bronchiolocentric cellular interstitial pneumonia, interstitial fibrosis, non-caseating epithelioid granulomas, epithelioid histiocytic infiltrate in the respiratory bronchioles and intraluminal budding fibrosis. The five cases with open lung biopsy performed also showed neutrophilic infiltrate in the alveoli. The two lung transplant cases were complicated by severe fibrotic changes. CONCLUSIONS: Chronic EAA demonstrates characteristic pathologic features. Definitive diagnosis requires correlation with clinical and radiologic findings due to possible morphologic mimicry of other diffuse parenchymal lung diseases.

[Effective analysis of the posterior vertebral pedicle screw fixation, vertebral body removal, decompression and titanium mesh reconstruction for the treatment of the lower lumbar fractures].

OBJECTIVE: To evaluate the effect of the treatment of the lower lumbar fractures by posterior vertebral pedicle screw fixation, vertebral canal decompression,bone graft and titanium mesh reconstruction. METHODS: From January 2006 to December 2008, 22 patients with lower lumbar fractures were treated by posterior vertebral pedicle screw fixation, vertebral canal decompression, bone graft and titanium mesh reconstruction at same period. There were 18 males and 4 females with an average age of 43.8 years ranging from 22 to 63 years old. The injured vertebrae were L3 in 11 cases, L4, in 8 cases, and L5 in 3 cases. The operative time, blood loss, the preoperative and postoperative vertebral height,sagittal index, and the lumbar lordosis angle were recorded and evaluated. RESULTS: The operative time was 3 to 4.2 hours (means 3.6 h). The blood loss averaged 1300 ml (900 to 1500 ml). The preoperative and postoperative sagittal index were (57.5 +/- 7.6)% and (93.5 +/- 8.1)%, respectively. The preoperative and postoperative lumbar lordosis angle were (34.3 +/- 7.3) degrees and (38.5 +/- 9.8) degrees, respectively. All patients were followed up for 10 months to 3 years (means 2.6 years). No fixation were failed,the segment of titanium mesh reconstruction obtained bone healing, no pseudoarticulation formation. At the last time of followed-up, 15 patients with nerve injuries were evaluated according to Frankel grade, there were 10 cases in grade E, 4 in D, 1 in C. According to the low back outcome scores (LBOS), the results were excellent in 20 cases, good in 1, fair in 1. CONCLUSION: The stability of the lower lumbar spine can be reconstructed by bone graft and titanium mesh combined with transpedicular screw fixation through a posterior approach. The decompression and vertebral body removal can also be performed in this approach. The recovery of the vertebral height and lumbar lordosis can prevent the delayed neurological deficit and traumatic kyphosis.

[Fixed-point release with small needle knife for the treatment of upper plexus thoracic outlet syndrome].

OBJECTIVE: To study therapeutic effects and mechanisms of fixed-point release with small needle knife for the treatment of upper plexus thoracic outlet syndrome. METHODS: Among 32 patients, 22 patients were female, and 10 patients were male, ranging in age from 25 to 55 years. The disease course ranged from one month to 3 years. All the patients were unilateral thoracic outlet syndrome. The painful trabs at the facet joints of C(5,6) and infraspinatus muscle were fixed-point released one to four times every week. RESULTS: All the patients relieved pain immediately after treatment. Among 26 patients with decreased muscle strength before operation, 20 patients got muscle strength improved after treatment immediately. Among 18 patients with touch and pain sensation compromised, 8 patients got the sensation improved after operation. All the patients were followed up for 1 year. According to Wood evaluation criterion, 19 patients got an excellent result, 7 good, 3 poor, and 3 bad. One patient was transferred to operation. There were no complications. CONCLUSION: Fixed-point release with small needle knife for the treatment of upper plexus thoracic outlet syndrome is safe and curative.

Tyrphostin AG1478 suppresses proliferation and invasion of human breast cancer cells.

Inhibition of epidermal growth factor receptor (EGFR) signaling is a promising treatment strategy for malignant tumors. In this study, we evaluated the effectiveness of tyrphostin AG1478, a potent and specific inhibitor of EGFR tyrosine kinase, on the growth, apoptosis and invasion of breast cancer cells. Western blotting demonstrated that AG1478 inhibited the phosphorylation of EGFR, ERK1/2 and AKT in a dose-dependent manner. Three proliferation analyses, MTT, cell counting, and clone formation assay, consistently showed that AG1478 significantly inhibited cell proliferation in a dose-dependent manner. FACS analysis demonstrated that AG1478 promoted cell apoptosis. In addition, TRAP assay exhibited that AG1478 significantly suppressed telomerase activity of tumor cells, which was parallel with growth inhibition. Semi-qantitative RT-PCR revealed that the suppression of telomerase activity was correlated with the decreased expression of human telomerase catalytic subunit (hTERT) mRNA, the rate-limiting determinant of its enzyme activity. These data suggest that AG1478 suppressed cellular growth by inhibiting cellular proliferation, inducing apoptosis and inhibiting telomerase activity. Furthermore, we also examined the effects of AG1478 on cellular invasion. Boyden chamber invasion assay showed that AG1478 significantly inhibited cell invasion in a dose-dependent manner. Western blotting revealed that AG1478 could down-regulate the expression of MMP-9, which may be one of the mechanisms by which AG1478 suppressed cellular invasion. In conclusion, this study demonstrated that Tyrphostin AG1478 effectively inhibited the proliferation and invasion of breast cancer cells. Tyrphostin AG1478 may be a potential EGFR-targeted therapeutic agent for breast cancer.

Expression of E-cadherin, beta-catenin, cathepsin D, gelatinases and their inhibitors in invasive ductal breast carcinomas.

BACKGROUND: E-cadherin, beta-catenin, cathepsin D, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 are all invasion-related proteins. The expression patterns of these proteins in invasive ductal breast carcinomas, and their associations with known clinicopathological parameters, tumor recurrence and expressions of estrogen receptor (ER), progesterone receptor (PR), PS2 and c-erbB2 were not well studied in Chinese patients. METHODS: In a set of 94 invasive ductal breast carcinomas, protein expressions of these molecular markers were investigated by immunohistochemistry, and their associations with known clinicopathological parameters, tumor recurrence and expressions of ER, PR, PS2 and c-erbB2 were also examined. In addition, the interrelationship between the expressions of these proteins were studied. RESULTS: Preserved membrane E-cadherin expression was associated with late tumor stage and tumor recurrence, whereas the reduced junctional beta-catenin associated with positive lymph node status and c-erbB2 overexpression. Positive staining of cathepsin D in tumor stromal cells displayed a significant association with late tumor stage. High expression of MMP-2 in cancer cells was associated with large tumor size and PR positive expression. TIMP-2 expression was positively associated with tumor recurrence. In addition, inter-relationship between the expressions of these biomarkers was also assessed. Cathepsin D staining in cancer cells was inversely correlated with its staining in stromal cells, and also inversely correlated with MMP-2 staining in tumor stromal cells. MMP-2 expression in stromal cells displayed an inverse correlation with TIMP-2 expression. MMP-9 expression displayed parallel associations with TIMP-1 and TIMP-2 expression. CONCLUSION: Evaluation of E-cadherin, beta-catenin, cathepsin D, MMP-2 and TIMP-2 expression may be of some help in more accurately predicting the prognosis of invasive ductal breast carcinomas.

Aberrant methylation of multiple genes in gastric carcinomas.

In this study, we examined aberrant methylation of the E-cadherin, estrogen receptor, RB1 , p16, p15, p14, and MGMT genes by the methylation-specific polymerase chain reaction method in 101 gastric carcinomas. Hypermethylation was detected in E-cadherin, estrogen receptor, RB1, p16, p14, p15, and MGMT at the rates of 27.7%, 44.6%, 44.6%, 30.7%, 19.2%, 7.7%, and 6.9%, respectively. A total of 82.2% cases had methylation in at least 1 of these genes, and 44.6% had methylation in 2 or more of these genes. Methylation of RB1 was associated with absence of lymph node metastasis. Methylation of estrogen receptor was associated with age and tumor location. Methylation of E-cadherin coincided with methylation of p16 or estrogen receptor. Moreover, loss of p16 protein was strongly associated with its gene methylation. This study indicates that aberrant methylation of multiple genes is involved in gastric carcinogenesis.

Effects of cotransfection of antisense-EGFR and wild-type PTEN cDNA on human glioblastoma cells.

The main molecular genetic changes identified in glioblastomas are overexpression/amplification of the epidermal growth factor receptor (EGFR) gene and mutation/ deletion of the tumor suppressor PTEN gene. These two genetic changes both play important roles in glial tumorigenesis and progression. In this study, we demonstrated that wild-type PTEN transfection inhibited the growth and transforming ability of U87MG cells by 69.3% and 73.5%, respectively. On the other hand, antisense-EGFR transfection inhibited the growth and transforming phenotype of these cells by 50.3% and 46.8%, respectively. However, cotransfection of U87MG cells with wild-type PTEN and antisense EGFR constructs could inhibit the cellular growth by 91.7%. The transforming phenotype of these cells was completely inhibited. In addition, these cotransfected cells showed a differentiated form and expressed much lower telomerase activity than cells transfected with wild-type PTEN or antisense-EGFR alone. In summary, these results suggest that cotransfection is a better approach to suppress glioma cell growth than wild-type PTEN transfer or antisense-EGFR transfection alone. This approach may prove useful as an adjunct therapy in the treatment of glioblastomas.

[Effect of antisense epidermal growth factor receptor cDNA transfection on telomerase activity of glioblastomas cells].

OBJECTIVE: To study the effects of antisense epidermal growth factor receptor (EGFR) cDNA transfection on telomerase activity and telomere length of glioblastomas U87MG cells and the mechanism. METHODS: Glioblastoma U87MG cells, which over-expressed EGFR, were transfected with antisense-EGFR cDNA constructs. Several clones stably expressing lower or undetectable levels of EGFR protein were obtained. The effect of antisense-EGFR cDNA on telomerase activity was assessed by Telomeric Repeat Amplification Protocol (TRAP) assay. The effect of antisense-EGFR cDNA on telomere length was determined by Southern blot hybridization. The mRNA expressions of hTERT, hTEP1 and c-myc were analyzed by semi-quantitative PCR. RESULTS: U87MG cells that were transfected with antisense-EGFR cDNA expressed lower level of EGFR and were less responsive to the growth stimulation of EGF compared with the control cells. Telomerase activity was significantly decreased in the antisense-EGFR clones. Telomere length was shortened. The mRNA expression of hTERT was slightly decreased in the antisense-EGFR clones, whereas the expressions of hTEP1 and c-myc were not altered. CONCLUSION: Antisense-EGFR cDNA transfection can sufficiently inhibit EGFR signal transduction pathway, decrease telomerase activity and shorten telomere length, which may be a new mechanism of antisense-EGFR approach in tumor suppression. The down-regulation of hTERT mRNA may contribute to the decreased telomerase activity in the antisense-EGFR clones.