Association of liver abnormalities with in-hospital mortality in patients with COVID-19.

BACKGROUND & AIMS: The evolution and clinical significance of abnormal liver chemistries and the impact of hepatitis B infection on outcome in patients with COVID-19 is not well characterized. This study aimed to explore these issues. METHODS: This large retrospective cohort study included 2,073 patients with coronavirus disease 2019 (COVID-19) and definite outcomes in Wuhan, China. Longitudinal liver function tests were conducted, with associated factors and risk of death determined by multivariate regression analyses. A prognostic nomogram was formulated to predict the survival of patients with COVID-19. The characteristics of liver abnormalities and outcomes of patients with COVID-19, with and without hepatitis B, were compared after 1:3 propensity score matching. RESULTS: Of the 2,073 patients, 1,282 (61.8%) had abnormal liver chemistries during hospitalization, and 297 (14.3%) had a liver injury. The mean levels of aspartate aminotransferase (AST) and direct bilirubin (D-Bil) increased early after symptom onset in deceased patients and showed disparity compared to levels in discharged patients throughout the clinical course of the disease. Abnormal AST (adjusted hazard ratio [HR] 1.39; 95% CI 1.04-1.86, p = 0.027) and D-Bil (adjusted HR 1.66; 95% CI 1.22-2.26; p = 0.001) levels at admission were independent risk factors for mortality due to COVID-19. A nomogram was established based on the results of multivariate analysis and showed sufficient discriminatory power and good consistency between the prediction and the observation. HBV infection in patients did not increase the risk of poor COVID-19-associated outcomes. CONCLUSIONS: Abnormal AST and D-Bil levels at admission were independent predictors of COVID-19-related mortality. Therefore, monitoring liver chemistries, especially AST and D-Bil levels, is necessary in hospitalized patients with COVID-19. LAY SUMMARY: Liver test abnormalities (in particular elevations in the levels of aspartate aminotransferase [AST] and direct bilirubin [D-Bil]) were observed after symptom onset in patients who went on to die of coronavirus disease 2019 (COVID-19). Abnormal levels of AST and D-Bil at admission were independent predictors of COVID-19-related mortality. HBV infection in patients did not increase the risk of poor COVID-19-associated outcomes.

Clinical course of patients on maintenance hemodialysis and COVID-19: a retrospective longitudinal study.

Coronavirus Disease 2019 (COVID-19) emerges as a global pandemic and there is a lack of evidence about the clinical course and outcome of patients on maintenance hemodialysis (MHD). Here we conducted a retrospective longitudinal study aimed to analyze the clinical features and outcome of MHD patients hospitalized with COVID-19. Of 3126 inpatients with COVID-19 at 3 Branches of Wuhan Tongji Hospital from Jan 18th to Mar 9th, 2020, 19 patients were undergoing maintenance hemodialysis. Among the 19 MHD patients with COVID-19, 6 patients (31.6%) died, and 13 patients (68.4%) were able to be discharged. Baseline characteristics, clinical courses, laboratory findings, and dynamic trajectories of major laboratory markers were compared between survivors and nonsurvivors. According to our findings, MHD patients with COVID-19 who experienced non-surviving outcome had more elevated CRP, IL6 and procalcitonin as well as fibrinogen levels at various points compared to survivors. Thus the dysregulation of immune response as well as coagulation abnormalities might be highly involved in the pathological process of COVID-19, contributing to the poor prognosis in MHD patients.

Efficacy of topical versus oral 5-aminosalicylate for treatment of 2,4,6-trinitrobenzene sulfonic acid-induced ulcerative colitis in rats.

5-aminosalicylic acid (5-ASA) is drug of choice for the treatment of ulcerative colitis (UC). In this study, the efficacy of topical versus oral 5-ASA for the treatment of UC was examined as well as the action mechanism of this medication. A flexible tube was inserted into the rat cecum to establish a topical administration model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced UC. A total of 60 rats were divided into sham operation group (receiving an enema of 0.9% saline solution instead of the TNBS solution via the tube), model group, topical 5-ASA group, oral Etiasa group (a release agent of mesalazine used as positive control) and oral 5-ASA group (n=12 each). Different treatments were administered 1 day after UC induction. The normal saline (2 mL) was instilled twice a day through the tube in the sham operation group and model group. 5-ASA was given via the tube in the topical 5-ASA group (7.5 g/L, twice per day, 100 mg/kg), and rats in the oral Etiasa group and oral 5-ASA group intragastrically received Etiasa (7.5 g/L, twice per day, 100 mg/kg) and 5-ASA (7.5 g/L, twice per day, 100 mg/kg), respectively. The body weight was recorded every day. After 7 days of treatment, blood samples were drawn from the heart to harvest the sera. Colonic tissues were separated and prepared for pathological and related molecular biological examinations. The concentrations of 5-ASA were detected at different time points in the colonic tissues, feces and sera in different groups by using the high pressure liquid chromatography (HPLC). The results showed that the symptoms of acute UC, including bloody diarrhea and weight loss, were significantly improved in topical 5-ASA-treated rats. The colonic mucosal damage, both macroscopical and histological, was significantly relieved and the myeloperoxidase activity was markedly decreased in rats topically treated with 5-ASA compared with those treated with oral 5-ASA or Etiasa. The mRNA and protein expression of IL-1ß, IL-6, and TNF-α was down-regulated in the colonic tissue of rats topically treated with 5-ASA, significantly lower than those from rats treated with oral 5-ASA or Etiasa. The concentrations of 5-ASA in the colonic tissue were significantly higher in the topical 5-ASA group than in the oral 5-ASA and oral Etiasa groups. It was concluded that the topical administration of 5-ASA can effectively increase the concentration of 5-ASA in the colonic tissue, decrease the expression of proinflammatory cytokines, alleviate the colonic pathological damage and improve the symptoms of TNBS-induced acute UC in rats.

Efficacy of gemcitabine plus platinum agents for biliary tract cancers: a meta-analysis.

The objective of this study was to carry out a meta-analysis of the efficacy of gemcitabine+platinum agent regimens in the treatment of advanced biliary tract cancer (BTC). PubMed and Google Scholar were searched using the following combination of search terms: gemcitabine, oxaliplatin, cholangiocarcinoma, biliary, gallbladder, bile duct. Studies were eligible for inclusion in the meta-analysis if they were randomized trials on the use of gemcitabine plus a platinum agent for the treatment of advanced (unresectable or metastatic cancer) BTC. Outcomes of interest were response rate, overall survival, and progression-free survival. Pooled odds ratios/differences in median survival and 95% confidence intervals (CIs) were determined for each outcome. A total of 47 records were identified in the initial search. Ultimately, three open-label randomized trials (two phase 2 and one phase 3) met the eligibility criteria and were included in the meta-analysis. Two studies compared gemcitabine plus cisplatin with gemcitabine alone, whereas the other study compared gemcitabine plus oxaliplatin with fluorouracil-folinic acid. The total number of patients in the studies ranged from 54 to 410. The overall analyses revealed that all survival outcomes assessed were significantly more favorable for patients treated with gemcitabine plus platinum agents than for patients not treated with this combination. Response rates: odds ratio=2.639, 95% CI=1.210-5.757, Z=2.439, P=0.015; pooled difference in median overall survival=3.822 months, 95% CI=1.798-5.845 months, Z=3.702, P<0.001; pooled difference in median progression-free survival=3.268 months, 95% CI=1.996-4.541 months, Z=5.035, P<0.001. Patients with advanced BTC who are treated with gemcitabine plus platinum agents may experience better survival outcomes compared with patients who are not treated with this combination of chemotherapy.

Effects of the 24 N-terminal amino acids of p55PIK on endotoxinstimulated release of inflammatory cytokines by HaCaT cells.

This study aimed to examine the effect of the 24 N-terminal amino acids (N24) of p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (PI3K), on the endotoxin lipopolysaccharide (LPS)-stimulated release of the cytokines (CKs) by HaCaT cells. The fusion protein, trans-acting activator of transcription (TAT)-N24 (an experimental peptide, EP) containing the N24 of PI3K-p55PIK, was constructed, and TAT-N24 fusion peptide was expressed and identified in BL21 E·coli. HaCaT cells (a human keratinocyte cell line) was cultured and stimulated by LPS at 100 ng/mL for 1, 2, 4, 8, 16 or 24 h, or by LPS at 10, 100 ng/mL, 1, 10 or 100 µg/mL of for 4 h. Changes in the protein and mRNA levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) released by HaCaT cells following EP intervention were determined by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Immunofluorescence confocal laser scanning microscopy was utilized to detect the protein expression and translocation of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB p65) in HaCaT cells. The expression of the NF-κB inhibitor alpha (IκB-α) protein in LPS-stimulated HaCaT cells after the EP intervention was measured by Western blotting. The results showed that EP treatment increased TNF-α secretion from HaCaT cells. EP at certain concentrations could effectively inhibit the LPS-stimulated release of TNF-α, IL-6 and IL-8 from HaCaT cells. The ELISA assay demonstrated that the concentrations of TNF-α, IL-6 and IL-8 in the supernatants of LPS-stimulated cells were reduced from 208.06±30.18, 86.4±9.78 and 260.59±54.05 pg/mL to 121.78±22.26, 53.18±7.36 and 125.08±35.17 pg/mL, respectively, in the supernatants of cells treated by LPS and EP combined. Real-time PCR also revealed that the expression of the three pro-inflammatory CKs was significantly decreased after EP intervention. Immunofluorescence confocal laser scanning microscopy showed that NF-κB p65 protein was primarily expressed in the cytoplasm of non-stimulated HaCaT cells. After LPS stimulation, NF-κB p65 was translocated into the nucleus, and the nuclear expression of this protein increased. The nuclear NF-κB p65 protein expression was inhibited after the addition of EP. Western blotting showed that IκB-α expression began to decrease 30 min after LPS stimulation and declined to a trough 4 h later. IκB-α expression began to gradually recover 16 h after LPS stimulation but remained at a lower-than-normal level at 24 h. Greater IκB-α expression was found in cells treated with LPS and EP combined than those treated with LPS alone. It was concluded that EP can effectively inhibit the LPS-stimulated expression of TNF-α, IL-6, and IL-8, which involves the inhibition of the hydrolysis of IκB-α and thereby blockage of the nuclear translocation of NF-κB p65.

Remarkably reduced expression of FoxO3a in metaplastic colorectum, primary colorectal cancer and liver metastasis.

The forkhead family members of transcription factors (FoxOs) are expected to be potential cancer-related drug targets and thus are being extremely studied recently. In the present study, FoxO3a, one major member of this family, was identified to be down-regulated in colorectal cancer through micro-array analysis, which was confirmed by RT-PCR and Western blot in 28 patients. Moreover, immunohistochemistry (IHC) showed that the expression levels of FoxO3a were remarkably reduced in 99 cases of primary colorectal cancer, liver metastasis, and even in metaplastic colorectal tissue. IHC also revealed an exclusion of FoxO3a from the nucleus of most cells of tumor-associated tissues. Silencing FoxO3a by siRNA led to elevation of G2-M phase cells. We conclude that the downregulation of FoxO3a may greatly contribute to tumor development, and thus FoxO3a may represent a novel therapeutic target in colorectal cancer.

[Expression of human papillomavirus type 16/18 in human cervical carcinomas by the quantum dot fluorescent in-situ hybridization].

OBJECTIVE: To investigate fluorescence in situ hybridization labeled with quantum dots (QDs) for the detection of human papillomavirus 16/18 (HPV16/18) infection in cervical carcinoma patients. METHODS: A total of 80 biopsy samples of squamous carcinoma of cervix were assayed for HPV 16/18 infection by using quantum dot labeled fluorescent in situ hybridization (QD-FISH) and chromogenic in situ hybridization (CISH) techniques, respectively. The results obtained by using two different methods were statistically analyzed. RESULTS: The positive rate for HPV16/18 by QD-FISH was 88.8% (71/80), higher than that (80.0%) by CISH, however, the result was statistically not significant (P=0.127). The positive detection rates for HPV16/18 by using both methods increased coincidentally with raising of the tumor grading stage. CONCLUSION: The sensitivity and specificity of HPV infection detectable by QD-FISH is higher than that by the CISH technique.

Expression of HIF-1α is a predictive marker of the efficacy of neoadjuvant chemotherapy for locally advanced cervical cancer.

Platinum-based, arterial infusion chemotherapy as a neoadjuvant chemotherapy (NACT) followed by hysterectomy may be efficient for the treatment of locally advanced cervical cancer and improve prognosis. It is important to predict whether the NACT would be effective before it is launched. Hypoxia inducible factor-1α (HIF-1α) is the master transcriptional regulator of the cellular response to altered oxygen concentration. HIF-1α protein expression is elevated in numerous human malignancies, contributes to poor disease outcome, and has been reported to induce tumorigenesis and chemoresistance. In the present study, patients with International Federation of Gynecology and Obstetrics stage IIB-IIIB cervical cancer (n=59) between 2008 and 2014 were assessed for HIF-1α expression by immunohistochemistry. Tumor samples were obtained by biopsy before any treatment. A double-path chemotherapy regimen, paclitaxel (intravenous) plus cisplatin (intra-arterial injection into the uterine region), was used as NACT. The patients were then separated into two groups according to NACT response: One group comprised patients with NACT, for whom the response to treatment was efficient resulting in complete/partial remission of the tumor (CR + PR group; n=52), the other group contained patients with NACT, for whom the result of the treatment was a stable/progressive disease (SD + PD group; n=7). HIF-1α expression was tested in paraffin-embedded sections using immunohistochemistry. HIF-1α expression was significantly higher in the SD + PD group compared with the CR + PR group (P=0.029). The overall survival time was significantly longer in the CR + PR group compared with the SD + PD group (P<0.001). When the patients were divided into two groups based on HIF-1α expression levels. Low (weighted score ≤4, n=39) and high (weighted score ≥6, n=20) expression level groups; the low HIF-1α expression group was significantly more susceptible to NACT treatment (P=0.025). Cox hazard analysis revealed that a high level of HIF-1α expression and lymph node metastases were significant independent predictors of poor overall survival (P=0.025, HR=6.354; P=0.020, HR=6.909, respectively). These results indicated that the expression of HIF-1α may be able to predict the efficiency of NACT and may be considered an independent prognostic factor for stage IIB-IIIB cervical cancer.

[Overexpression of hSav1 promotes Mst1-induced apoptosis in HeLa cells].

OBJECTIVE: To elucidate the effect of hSav1 expression on Mst1-mediated apoptosis in HeLa cells. METHODS: Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 micromol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay. RESULTS: Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSav1 could be detect from the anti-Mst1 immunoprecipitation complex. The immunofluorescent labeling showed that hSav1 and Mst1 had the same localization in cells. Overexpressed protein hSav1 did not induce a significant cell apoptosis. However, co-expression of hSav1 with Mst1 resulted in a significant increase of apoptosis above the level seen with Mst1 alone (24.5% +/- 2.4% vs. 39.3% +/- 4.0%, P < 0.05). CONCLUSION: Our findings indicate that hSav1 is a newly identified protein that interacts with Mst1 and augments Mst1-mediated apoptosis.

Detection of metastatic cancer cells in mesentery of colorectal cancer patients.

AIM: To detect the existence of isolated cancer cells in the mesentery of colorectum (named as Metastasis V), and investigate its clinical significance in colorectal cancer (CRC) patients. METHODS: Sixty-three CRC patients who received radical excision between January 2012 and September 2015 were included. All the patients underwent laparoscopy-assisted radical colorectomy or proctectomy [with complete mesocolic excision (CME) or total mesorectal excision (TME)] with R0 dissections at the Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The location and size of the primary lesions were recorded immediately after the tumor was removed, with the surrounding mesenterium completely separated along the intestinal wall. Each dissected mesentery sample was analyzed for hematoxylin-eosin staining and immunohistochemistry using cytokeratin 19 antibody. Image Pro Plus Software 6.0 (Media Cybernetics, CA, United States) was used to semi-quantitatively measure the concentration of the cytokeratin 19 immunohistochemistry. The correlation between metastasis found in mesentery and clinicopathological characteristics was examined. The prognosis of patients was also evaluated by preoperative serum CEA level. RESULTS: Metastasis V was detected in 14 of 63 (22.2%) CRC patients who underwent laparoscopy-assisted radical colorectomy or proctectomy (with CME or TME) with R0 dissection in our hospital between January 2012 and September 2015. There was no significant difference in age, gender, tumor size, and tumor location in patients with Metastasis V (P > 0.05). Metastasis V was more likely to occur in poorly differentiated tumor (5/11; 45.5%) than moderately (8/46; 17.4%) and well- differentiated one (1/6; 16.7%). The Metastasis V in N2 stage (9/14; 64.3%) was more frequent that in the N0 stage (3/35; 8.6%) or N1 stages (2/14; 14.3%). In addition, Metastasis V was positively related to the tumor invasive depth (T1:0/1, 0%; T2:1/12, 8.3%; T3:7/39, 17.9%; T4:6/11, 54.5%). Furthermore, preoperative serum CEA level in Metastasis V-positive patients was significantly higher than in Metastasis V-negative patients (4.27 ng/mL vs 3.00 ng/mL). CONCLUSION: Metastasis V might be associated with a poor prognosis of CRC patients.

miR-124 modulates gefitinib resistance through SNAI2 and STAT3 in non-small cell lung cancer.

Gefitinib is used as a first-line treatment for advanced non-small cell lung cancer (NSCLC). Unfortunately, most NSCLC patients inevitably develop gefitinib resistance during treatment. In addition to EGFR mutation status, the mechanisms involved are largely unknown. In this study, we showed that miR-124, a tumor suppressor, was significantly down-regulated in gefitinib-resistant NSCLC patients and cell lines compared with gefitinib-sensitive patients and cell lines. In addition, the miR-124 depletion induced gefitinib resistance, and miR-124 overexpression sensitized gefitinib-resistant cells to gefitinib. Mechanistic analysis revealed that miR-124 decreased SNAI2 and STAT3 expression by directly targeting their 3'UTRs and that knocking down SNAI2 or STAT3 partly reversed the gefitinib resistance induced by miR-124 depletion. Our data demonstrate that the miR-124 plays a new critical role in acquired resistance to gefitinib and that the manipulation of miR-124 might provide a therapeutic strategy for reversing acquired gefitinib resistance.

Suicide gene therapy of hepatocellular carcinoma and delivery procedure and route of therapeutic gene in vivo.

OBJECTIVE: To study the induction of sensitivity to ganciclovir (GCV) or acyclovir (ACV) in human hepatocellular carcinoma (HCC) cell line transferred by an Epstein-Barr virus (EBV)-based replicon expression vector carrying the herpes simplex virus thymidine kinase (HSV-tk) gene, including killing and "bystander" effect, and also the gene delivery procedure and route of gene therapy in vivo for HCC. METHODS: Liposome-entrapped plasmid pDR2/tk was transferred into HCC cells, and then different concentrations of GCV or ACV were added. The transferred cells were mixed with untransferred HCC cells in different proportion and 200 micromol/L GCV was then added into each well. After 72 hours, all samples were measured by MTT colorimetric assay. An EBV-based plasmid eukarotic expression vector carrying IL-2 cDNA was used. Three models of gene direct injection in the local liver, injection through the portal vein, and injection through the embolized hepatic artery were established in closed Wister rats. For each model, two subgroups, injected either naked plasmid DNA or lipofectin-plasmid complex were included. The expression of the IL-2 gene was regularly examined immunohistochemically. RESULTS: GCV or ACV could apparently kill the transferred HCC cells at a concentration of 0.2 micromol/L. The inhibition rate was changed with different drug concentrations. The "bystander" effect was obviously induced at a transferred to untransferred HCC cells ratio of 1:5. IL-2 gene expression was observed in liver cells of all animals on day 3, which reached peak within 3-7 days, and declined after day 7. Injection of naked plasmid DNA through the hepatic artery plus embolization obtained a best expression. CONCLUSIONS: EBV-based vector is suitable for carrying suicide gene therapy for hepatocellular carcinoma. Gene direct delivery in vivo combined with interventional surgery can be used to treat hepatocellular carcinoma.

Expression of Cav-1 in tumour cells, rather than in stromal tissue, may promote cervical squamous cell carcinoma proliferation, and correlates with high-risk HPV infection.

Altered expression of caveolin-1 (Cav-1) is observed in various types of cancers. However, little research has been reported regarding the correlation between the expression of Cav-1 and cervical cancer. Here, we investigated the clinical significance of Cav-1 expression using quantum dot (QD)-based immunofluorescence staining in cervical cancer and its correlation with high-risk human papilloma virus (HPV) infection detected by chromogenic in situ hybridization. Our results showed that the positive rates of Cav-1 protein in normal cervical mucosa, CIN, cervical adenocarcinoma and SCC were: 0, 33, 19 and 55%, respectively. The differences in Cav-1 protein expression in cervical SCC compared to the other three groups were all statistically significant. Absence of stromal Cav-1 protein in 58 cases of cervical SCC was 67%. The positive rates of the Cav-1 protein in tumour and stromal cells of cervical SCC were not correlated with clinicopathological parameters. In the cervical SCC tissues, Cav-1 expression in tumour cells was not associated with stromal Cav-1 expression, but a positive correlation existed with the PCNA protein and high-risk of HPV infection. The results presented here suggest that expression of Cav-1 in the tumour cells, rather than in the stromal tissue surrounding the tumour, may promote cervical SCC cell proliferation, and correlates with high-risk HPV infection.

[Inhibitory effect of N-terminal 24 amino acids of the p55 gamma, a regulatory subunit of phosphoinositide 3-kinase, on proliferation of colon carcinoma cell line HT29].

BACKGROUND & OBJECTIVE: p55 gamma is one of the regulatory subunits of phosphoinositide 3-kinase (PI3K), which plays an important role in the regulation of PI3K activity. This study was to explore the inhibitory effect of the N-terminal 24 amino acids of the p55 gamma on proliferation of colon carcinoma cell line HT29. METHODS: Ad-N24p55-GFP, containing N-terminal 24 amino acids of the p55 gamma, and control vector Ad-GFP were constructed, and used to infect HT29 cells. The cell cycle progression was detected using flow cytometry. DNA synthesis was analyzed using the BrdU/PI method. The nude mice model bearing HT29 xenograft tumors was used to study the effect of Ad-N24p55-GFP against the tumor. RESULTS: Compared with cells infected with Ad-GFP, cells infected with Ad-N24p55-GFP at the G0/G1 phase were increased from 65.11% to 73.39% P < 0.05, and cells at S and G2/M phases were decreased from 17.37% to 15.08% and from 17.51% to 11.13%, respectively; BrdU-positive cells were decreased from 24.82% to 9.27%. In the nude mice model, the tumor growth was inhibited after the administration of Ad-N24p55-GFP. The tumor weight was (0.32+/-0.08)g vs. (10.67+/-0.3)g and (0.72+/-0.28)g in the Ad-N24P55-GFP group,the blank control group and the Ad-GFP group, respectively. CONCLUSION: In HT29 cells, overexpression of the N-terminal 24 amino acids of the p55 gamma can induce cell cycle arrest, inhibit DNA synthesis and inhibit tumor growth in the nude mice model of HT29 xenograft tumors.

[Preparation of suspension of gastric mucous membrane single cell and expression of cyclins in cells].

OBJECTIVE: To explore a method which can remove the gastric mucus in order to prepare mucous membrane single cell suspension for the research of cytomics. METHODS: Enzymology was used to remove the mucus gel and to separate mucous layer from the normal fresh gastric tissue. The mucous layer was broken to prepare single cell suspension with machine method. Expression of major cyclins in mucous layer cells was examined by cytoimmunochemistry, flow cytometry(FCM) and confocal microscopy. RESULTS: The 0.1% pepsin could dissolve the mucus gel and 1.2-2.4 U/L dispase could separate the mucous layer completely. The single mucous cell suspension was prepared successfully. FCM results from mucous single cell suspension revealed that expression of cyclin D(3), B(1) was obvious, that of cyclin D(2) was weak and that of cyclin D(1), A, E was the least. Similar results were found with confocal microscopy. CONCLUSIONS: Single cell suspension from mucous layer can be easily prepared by pepsin and dispase. Cyclins schedule expression in vivo is different from cyclins schedule expression in vitro.

[Correlations of GRIM-19 and its target gene product STAT3 to malignancy of human colorectal carcinoma].

BACKGROUND & OBJECTIVE: GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) gene is a specific protein to inhibit signal transducers and activators of transcription 3 (STAT3). STAT3 and its pathway are involved in modulating cell proliferation, apoptosis, differentiation, and mediating malignant transformation of cells. This study was to investigate the expression of GRIM-19 and its target gene STAT3 in human colorectal carcinoma tissues, and explore their roles in the tumorigenesis of colorectal carcinoma. METHODS: The expression of GRIM-19, STAT3 and its activated form p-STAT3 in 40 specimens of colorectal carcinoma, adjacent tissue, and normal tissue was determined by immunohistochemistry and Western blot. The correlations of the expression of GRIM-19, STAT3, and p-STAT3 to various clinicopathologic characteristics of colorectal carcinoma were analyzed statistically. The mRNA expression and gene mutation of GRIM-19 in colon cancer cell line SW480 and 23 specimens of colorectal carcinoma, adjacent tissue, and normal tissue were detected by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. RESULTS: The expression of both STAT3 and p-STAT3 were up-regulated in colorectal carcinoma. The mRNA and protein expression of GRIM-19 was obviously lower in colorectal carcinoma than in normal tissues. The expression of GRIM-19 was correlated to clinical stage and cell differentiation of colorectal cancer (P< 0.05). GRIM-19 expression in colorectal cancer was negatively correlated to STAT3 and p-STAT3 expression (Chi2 = 9.95, P = 0.00; Chi2 = 5.10, P = 0.02). No mutation of GRIM-19 gene was detected in colorectal carcinoma tissues. CONCLUSIONS: The low expression or absence of GRIM-19 may play an important role in the tumorigenesis of colorectal carcinoma. The high expression of STAT3 and the low expression of GRIM-19 co-exist in colorectal carcinoma, and may be related to malignant transformation and abnormal proliferation of cells.

[DNA damage and apoptosis of human airway epithelial cell lines caused by cigarette smoke extract].

BACKGROUND & OBJECTIVE: Cigarette smoke exposure has been reported to induce DNA damage in a variety of cell types. This study was to investigate DNA damage and apoptosis in normal human bronchial epithelial cell line NHBE and lung carcinoma cell line SPC-A1 caused by cigarette smoke extract. METHODS: NHBE and SPC-A1 cells were incubated with different concentrations of cigarette smoke extract. Cell viability was evaluated by MTT assay. Fluorescence-labeled anti-histone gamma-H2AX polyclonal antibody was used to detect DNA double-strand breaks (DSBS) in chromatin. DNA damage was analyzed by flow cytometry. The expression of gamma-H2AX was detected by Western blot. Cigarette smoke extract-induced cell apoptosis was detected by sub G1 peak method and annexin V-FITC/propidium iodide (PI) staining assay. Cell morphology of DNA damage and apoptosis was observed by confocal laser microscopy. RESULTS: MTT assay results showed that cigarette smoke extract decreased the viability of NHBE and SPC-A1 cells in time-and concentration-dependent manners. Cigarette smoke extract induced DSBS in NHBE cells and SPC-A1 cells, and led to H2AX phosphorylation (denoted gamma-H2AX) in time-and concentration-dependent manners. The maximal value of gamma-H2AX was seen about 4 h after the treatment, and the value decreased subsequently, but did not reduce to a normal level. Cell apoptosis appeared about 12 h after DNA damage. Cigarette smoke extract also initiated the accumulation of gamma-H2AX in these cells, and cell apoptosis morphology was observed 4 h later. CONCLUSION: Cigarette smoke extract can induce DSBS and apoptosis in NHBE and SPC-A1 cells in time-and concentration-dependent manners.

[Inducement of tumor cell autophagy and cell cycle analysis].

BACKGROUND & OBJECTIVE: Autophagy is the main phenomenon of type II programmed cell death which is also named as autophagic cell death, and autophagy has a close relationship with autophagic cell death. The relationship of apoptosis and cell cycle has been explored deeply, but little is known about the relationship of autophagic cell death and cell cycle. This study was to observe the correlation of autophagy induced by different methods to cell cycle. METHODS: Exponentially growing HeLa and SW480 cells, and peripheral blood lymphocytes (PBLs) from healthy donors, with or without 48 h stimulation of phytohemagglutinin (PHA), were treated with Hanks' solution (to produce starvation) or vincristine. Confocal laser microscope and transmission electron microscope (TEM) were used to detect autophagy; flow cytometry (FCM) was innovatively used to detect the cell cycle of autophagic cells with dipl-parameters of microtubule-associated protein 1 light chain 3 (MAP1-LC3-II)/PI. RESULTS: Autophagy of HeLa and SW480 cells induced by starvation or vincristine was observed in G1, S, and G2/M phases and increased along with the inducement time; no autophagy was observed in unstimulated PBLs. The positive rate of LC3-II, indicating the occurrence of autophagy, was lower than 2.62% when induced by starvation in Hanks' solution for 48 h, or 6.16% when induced by vincristine for 48 h. After PBLs were stimulated into cell cycle by PHA, autophagy was markedly detected 2 h after the indicated inducements. CONCLUSIONS: MAP1-LC3-II/DNA dipl-parameter analysis by FCM is a convenient and reliable method for simultaneously analyzing autophagy and cell cycle. Autophagy could be induced when cells are in cell cycle, while the cells in G0 phase are insensitive to the inducers.

[Promotive effect of decreased cyclin E threshold on cell proliferation].

BACKGROUND & OBJECTIVE: Many studies showed that high expression of Cyclin E promotes cell proliferation, but contrary data was also reported that cell proliferation didn't decrease with low expression of Cyclin E. In addition, we observed that many tumor cells have strong capability of proliferation with low expression of Cyclins, including Cyclin E. This study was to analyze effect of reduced Cyclin E threshold on proliferation of acute lymphocyte leukemia cell line MOLT-4 to explain the above phenomena. METHODS: We have established the model of decreased Cyclin E threshold in MOLT-4 cells by treating cells with low concentration (5 mmol/L) of caffeine for 2, and 4 h. The positive rates of proliferation cell nuclear antigen (PCNA), Ki67, and DNA strand break induction by photolysis (SBIP) were analyzed by flow cytometry. RESULTS: MOLT-4 cells presented sharply decrease of Cyclin E threshold, and increase of positive rates of PCNA, Ki67, and SBIP after treated with caffeine, especially at 2-h point. CONCLUSIONS: Decrease of Cyclin E threshold was accompanied by increase of cell proliferation. MOLT-4 cells may remain high proliferation capability with low level of Cyclin expression.