-
China World J Gastroenterol. ; 11:2714-2719.
-
Wenjuan Yu,
Leiming Wang,
Yuewei Wang,
Xiaoyan Xu,
Pengcheng Zou,
Miaozi Gong,
Jie Zheng,
Jiangfeng You,
Hua Wang,
Fang Mei, Fei Pei
[show abstract]
[hide abstract]
ABSTRACT: LASS2/TMSG1 was a novel tumor metastasis suppressor gene which was firstly cloned by our laboratory from non-metastatic and metastatic cancer cell variants of human prostate carcinoma PC-3M using mRNA differential display in 1999. LASS2/TMSG1 could interact with the C subunit of vacuolar ATPase (V-ATPase, ATP6V0C) and regulate V-ATPase activity. In an attempt to provide molecular mechanism of the interaction between LASS2/TMSG1 and V-ATPase, we constructed four variant transfectants containing different functional domain of LASS2/TMSG1 and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential. Results showed there were no obvious differences of V-ATPase expression among different transfected cells and the control. However, V-ATPase activity and Intracellular pH (pHi) was significantly higher in the variant transfectants with Homeodomain of LASS2/TMSG1 than that in the control using the pH-dependent fluorescence probe BECEF/AM. Immunoprecipitation, immunofluorescence and immuno-electron microscope alone or in combination demonstrated the direct interaction of Homeodomain of LASS2/TMSG1 and ATP6V0C. Loss of Homeodomain markedly enhanced the proliferation ability but weakened the apoptotic effect of LASS2/TMSG1 in PC-3M-1E8 cells. These lines of results for the first time contribute to the conclusion that LASS2/TMSG1 could regulate V-ATPase activity and intracellular pH through the direct interaction of its Homeodomain and the C subunit of V-ATPase. Their interaction could play impotant roles in the apoptosis of tumor cells. J. Cell. Biochem. © 2012 Wiley Periodicals, Inc.
Journal of Cellular Biochemistry 09/2012; · 2.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 (TMSG1), is a newly found tumor metastasis suppressor gene in 1999. Preliminary studies showed that it not only suppressed tumor growth but also closely related to tumor metastasis, however, its molecular mechanisms is still unclear. There have been reported that protein encoded by LASS2/TMSG-1 could directly interact with the C subunit of Vacuolar ATPase (V-ATPase), which suggested that LASS2/TMSG1 might inhibit the invasion and metastasis through regulating the function of V-ATPase. Thus, in this study, we explored the effect of small interference RNA (siRNA) targeting LASS2/TMSG1 on the invasion of human prostate carcinoma cell line PC-3M-2B4 and its molecular mechanisms associated with the V-ATPase. Real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blot revealed dramatic reduction of 84.5% and 60% in the levels of LASS2/TMSG1 mRNA and protein after transfection of siRNA in PC-3M-2B4 cells. The V-ATPase activity and extracellular hydrogen ion concentration were significantly increased in 2B4 cells transfected with the LASS2/TMSG1-siRNA compared with the controls. The activity of secreted MMP-2 was up-regulated in LASS2/TMSG1-siRNA treated cells compared with the controls; and the capacity for migration and invasion in LASS2/TMSG1-siRNA treated cells was significantly higher than the controls. Thus, we concluded that silencing of LASS2/TMSG1 may promote invasion of prostate cancer cell in vitro through increase of V-ATPase activity and extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2.
Journal of Cellular Biochemistry 07/2012; 113(7):2356-63. · 2.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Phosphoprotein associated with glycosphingolipid microdomains 1 (PAG) is an important negative regulator of immune signaling in T lymphocytes. However, newly emerging evidence has indicated that PAG may play important roles in tumor cells. Our previously reported cDNA microarray experiments identified PAG as a gene down-regulated in the high metastatic potential prostate cancer cell line PC-3M-1E8. In this study, we investigated the role of PAG in the proliferation, invasion and metastasis of prostate cancer cells and the underlying mechanisms. We confirmed that the expression of PAG at both the mRNA and protein levels was low in PC-3M-1E8 and DU145 cells compared to low metastatic potential prostate cancer cells PC-3M-2B4. In addition, we demonstrated that the reintroduction of PAG to PC-3M-1E8 and DU145 cells led to reduced proliferation through cell cycle arrest, decreased anchorage-independent growth and reduced invasion ability of tumor cells in vitro. This is the first report demonstrating that PAG inhibits the proliferation and invasion potential of prostate cancer cells via the interaction with RasGAP to recruit RasGAP to the cell membrane, where RasGAP hydrolyzes GTP to GDP, reduces the level of activated Ras, and ultimately suppresses the activation of ERK1/2, cyclin D1 and other effectors of the Ras signaling pathway. Morphologically, we observed that PAG could diminish the formation of pseudopodia on the cell surface and redistribute the intracellular F-actin in PC-3M-1E8 cells, which directly leads to the decreased invasion and metastasis potential of tumor cells. Taken together, these results suggest that PAG acts to inhibit the development and metastasis of prostate cancers and represents a novel therapeutic target for prostate cancer.
Oncology Reports 06/2012; 28(2):606-14. · 1.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To explore the effect of small interference RNA (siRNA) targeting homosapiens longevity assurance homologue 2(LASS2, or TMSG1) on the invasion of PC-3M-2B4 (a variant subline of human prostate carcinoma cell line PC-3M with low metastatic potential) and its molecular mechanisms.
PC-3M-2B4 cells were transfected with siRNA by using lipofectamine 2000. The expression of LASS2 mRNA and protein was detected after transfection by real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blot to screen the effective siRNA fragment. The V-ATPase activity of PC-3M-2B4 cells was detected by V-ATPase activity assay kit. The concentration of extracellular hydrogen ion was measured by pH-sensitive fluorescence probe bis-carboxyethyl-carboxyfluorescein (BCECF). The matrix metalloproteinase-2 (MMP-2) protein in the supernatant and cells was analyzed by Western blot. The activity of MMP-2 was examined by Gelatin zymography. Furthermore, the migration and invasion of cells were evaluated by in vitro wound migration assay and invasion assay.
RFQ-PCR and Western blot revealed dramatic reduction (84.5% and 60% ) in the levels of LASS2 mRNA and protein after transfection of siRNA-2 in PC-3M-2B4 cells. The V-ATPases activity and extracellular hydrogen ion concentration were significantly increased in PC-3M-2B4 cells transfected with the siRNA-2 compared with other control groups (P<0.05); There were no differences in the expression and secretion of MMP-2 protein between LASS2-siRNA treated cells and other control groups. However, the activity of MMP-2 was up-regulated in LASS2-siRNA treated cells compared with other control groups( P<0.05); and the capacity for migration and invasion in LASS2-siRNA treated cells was significantly higher than in other control groups (P<0.05).
Silencing of LASS2 can promote invasion of prostate cancer cells in vitro through the increase of the V-ATPases activity, extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2, indicating that LASS2 is a novel tumor metastasis suppressor gene.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 12/2011; 43(6):814-9.
-
[show abstract]
[hide abstract]
ABSTRACT: The tumor metastasis suppressor gene-1 (tmsg-1) was first cloned as a new tumor suppressor gene in our laboratory several years ago. Since then, however, despite the substantial progression that has been made in investigation of the biologic roles played by this gene, the manner in which it exerts its regulatory influence is still unknown. With transfection of various deletion or mutation constructs, we identified a potential enhancer and three potential silencers in the 5'-flanking region. However, it was particularly interesting to find that a region (+59 to +123 bp) of exon 1 exhibited a strong role in initiation of tmsg-1 gene transcription. Deletion of this region led to essentially complete loss of driving activity of exon-1 sequence on luciferase. Further analysis showed that transcription factors KLF6 and Sp1 are able to interact with each other and bind to their elements in this region. Co-transfection of pGL3-114/+123 with KLF6- and/or Sp1-expressing plasmids resulted in an elevation of luciferase activity and transcription level of tmsg-1, which was abolished by knockdown of KLF6 or Sp1. Analysis of metastatic capacity showed that cells with high metastatic capability exhibited a lower level of KLF6/TMSG-1 proteins with higher invasive capability and vice versa. Thus, we concluded that interaction of KLF6 and Sp1, together with their binding of elements in exon 1 are critical events in initiation of transcription of the tmsg-1 gene. These results reveal a hitherto unreported mechanism for initiation of transcription of the tmsg-1 gene.
Journal of Cellular Biochemistry 09/2011; 113(1):329-39. · 2.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1.
Luciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1. Co-immunoprecipitation (CoIP) was performed to analyze the interaction between KLF6 and Sp1. TMSG-1 and wt-KLF6 mRNA expressions in cells with different metastatic capacities were quantitated by real-time PCR. Cell invasive capability was determined by Matrigel invasion assay.
A 63 bp inducible regulatory region (+59 bp - +123 bp) in exon 1 was identified by luciferase assay using reporter plasmids with a series of TMSG-1 regulatory region deletions. Mutations in KLF6/Sp1 binding sites of this region resulted in a decrease of luciferase activity, while cotransfection with KLF6 or Sp1 expressing plasmids led to a remarkable increase of luciferase activity. EMSA and ChIP demonstrated that KLF6 as well as Sp1 interacted with this region. CoIP also indicated a possible interaction between KLF6 and Sp1 proteins. In the highly metastatic cell sublines, a low level of wild type KLF6 was associated synchronously with a low TMSG-1 level. Prostate carcinoma cells overexpressing KLF6 exhibited a higher TMSG-1 level and a lower invasive capability.
Transcription factor complex of KLF6 and Sp1 may participate in the inducible transcriptional regulation of TMSG-1, and a decreased wild type KLF6 expression is likely associated with a low TMSG-1 level in the highly metastatic cell sublines.
Zhonghua bing li xue za zhi Chinese journal of pathology 08/2011; 40(8):542-8.
-
[show abstract]
[hide abstract]
ABSTRACT: Our previous study showed that the interleukin-17 (IL-17) concentration in lung tissue and in bronchoalveolar lavage fluid (BALF) of rats with tobacco-smoke-induced chronic obstructive pulmonary disease (COPD) was higher than that of control group. However, whether IL-17 inhibitor could decrease the effect of tobacco smoking is not known yet. Objectives: To investigate the significance of IL-17 antibodies (Ab) in tobacco-smoke-exposed (TSE) mice.
Male C57/BL6 mice were randomly divided into three groups: TSE group, TSE + anti-IL-17 Ab group, and control group. The number of cells in BALF and the concentrations of IL-17, IL-6, IL-8 and MUC5AC in BALF and lung tissue homogenate were measured. Pulmonary function was measured by pressure sensors, and histologic analysis of the lungs was done in each group.
Lung function tests in TSE + anti-IL-17 Ab group were the same compared with TSE group (P > 0.05). The total cell count and the number of neutrophil cells in BALF were significantly higher in TSE group than the normal control group (P < 0.01). Compared with the TSE group, the total cell count in TSE + anti-IL-17 Ab group was decreased, and the percentage of neutrophils in BALF was highly decreased (P < 0.01). Airway inflammation was alleviated in TSE + anti-IL-17 Ab group by histologic analysis. The concentrations of IL-17 in lung tissue were significantly lower in TSE + anti-IL-17 Ab group than in TSE group (P < 0.01). IL-17 was mainly expressed in the epithelial cells in the airways of TSE mice. The concentration of IL-6, IL-8 and MUC5AC in BALF was decreased in TSE + anti-IL-17 Ab group compared with TSE group.
These data support a potential role for IL-17 in airway neutrophilic inflammation in TSE mice. Anti-IL-17 decreased the number of neutrophils as well as the concentration of MUC5AC in the BALF and attenuated neutrophilic airway inflammation.
Inhalation Toxicology 03/2011; 23(4):212-8. · 1.92 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the mechanism of influence of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behavior of human prostatic cancer cell line PC-3M-1E8.
The expression of GST-Raf1-RBD recombinant protein, a specific binding domain of active Ras (GTP-Ras), was induced by IPTG in JM109 bacterium. SDS-PAGE and coomassie brilliant blue staining were performed using cleaved product of the bacterium to determine the expression of the fusion protein. GST-pull down essay was designed to detect the level of active Ras in PGA1 and vector transfected, respectively and in the native PC-3M-1E8 cells. Western blotting was used to detect the expression levels of downstream proteins of Ras signal pathway which may be related to the function of PAG1. Phalloidine labeled by tetramethylrhodamine-5-(and-6) isothiocyanate (TRITC) was used for the staining of intracellular F-actin, Laser passing confocal microscopy was adopted for observing change of the cell morphology and the arrangement of F-actin.
After IPTG induction, the GST-Raf1-RBD recombinant protein, with a molecular weight of 33 000, was noticed to be highly expressed in JM109 bacterium. GST-pull down assay revealed that the expression level of active Ras markedly decreased after PAG1-transfection while the total Ras remained unchanged. The expression of p-ERK and cyclin D1 in the PAG1-transfected cells decreased in accordance with the level of active Ras. However, the expression of p21(WAF1/CIP1) and p-Akt didn't show any variation. Additionally, the structure of F-actin was turbulent and the pseudopodia of cells diminished conspicuously after PAG1-transfection. There was a high expression of PAG1 in normal human prostate tissue, however, the positive rate of PAG1 immuno-staining decreased in cases of prostatic adenocarcinoma, correspondent with increasing of the grading index of cell differentiation established in the Gleason grading system for the diagnosis of prostate adenocarcinoma.
An over-expression of PAG1 in PC-3M-1E8 cells effectively suppresses the activation of Ras and ERK, as well as the cyclin D1 expression, leading to an inhibition of the proliferation ability of tumor cells. The turbulence of F-actin and reduction of pseudopodia of cells result in an impairment of cell mobility, invasiveness and metastatic capability. In human prostate and prostatic adenocarcinoma tissues, the expression of PAG1 is related to the cellular differentiation and malignancy.
Zhonghua bing li xue za zhi Chinese journal of pathology 09/2010; 39(9):615-20.
-
[show abstract]
[hide abstract]
ABSTRACT: TMSG-1 is a newly discovered tumor metastasis suppressor gene, which plays important roles in promoting apoptosis and inhibiting invasion and metastasis of tumor cells. The inhibitory function of TMSG-1 in tumor cells may be related to vacuolar H+-ATPase and ceramide, but the underlying mechanism remains unknown. Studies on TMSG-1 are limited worldwide, and only a research group in Shanghai and our group have recently studied on it. As a new research field, the function of TMSG-1 remains to be explored. This review discusses the discovery of TMSG-1, structure of its encoded protein, its roles and possible mechanism in inhibiting tumor invasion and metastasis.
Chinese journal of cancer 07/2010; 29(7):697-702.
-
[show abstract]
[hide abstract]
ABSTRACT: To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro.
Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential)and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1, which was markedly downregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation. Real-time PCR and Western blot were used to confirm the gene expression of PAG1 at mRNA and protein levels. Full-length coding sequence of human PAG1 was subcloned into plasmid pcDNA3.0 and the recombinant plasmids were stably transfected into PC-3M-1E8. The cell proliferation ability, anchorage-independent growth, cell cycle distribution, apoptosis rates and invasive ability were detected by MTT, and in addition, soft agar colony formation, flow cytometry analysis and matrigel invasion assay using Boyden chamber were also carried out respectively. All experiments contained pcDNA3.0-PAG1-transfected clones, vector transfected clones and non-transfected parental cells.
A total of 327 differentially expressed genes were obtained between the high and low metastatic sublines of PC-3M cells, including 123 upregulated and 204 downregulated genes in PC-3M-1E8. A total of 281 genes, including 167 upregulated and 114 downregulated genes were obtained in PG-BE1 cells. Nine genes were simultaneously downregulated and 8 genes were upregulated in both high metastatic cell lines of PC-3M and PG. The expression of PAG1 at mRNA and protein level were decreased in the high metastatic subline PC-3M-1E8. Western blot revealed that PAG1 protein was downregulated in PC-3M-1E8 cell line which was in agreement with the gene expression at mRNA level. The proliferation ability and clonogenicity of PAG1 overexpression cells by stable transfection were markedly decreased in comparing with that of the control cells (P < 0.05). Colonies formed in stably PAG1-transfected cells, the vector-transfected clones and parental cells were 26.7 ± 5.2, 47.2 ± 3.2 and 52.3 ± 3.4 respectively (P < 0.05). Flow cytometry analysis showed that the stable PAG1-transfected cells at G₀-G₁ phase were significantly more than that of the control cells (P < 0.05). However, no difference of the apoptosis rate was found between PAG1-transfected cells and control cells (P > 0.05). The number of cells passing through the matrigel and multipore membrane was also decreased in the stable PAG1-transfected cells (35.1 ± 4.9) compared with those of the vector-transfected clones (127.6 ± 6.6) and parental cells (135.0 ± 5.0, P < 0.05).
Using cDNA microarray technique and differential gene expression analysis of sublines of the parental cancer cell lines enable of revealing the metastasis-related genes, among which PAG1 represents one of those under-expressed genes in the high metastatic subline PC-3M-1E8. Transfection expression of PAG1 suppresses cell proliferation, anchorage-independent growth and invasive ability of PC-3M-1E8 cells in vitro. Conclusively, PAG1 may play an important role in inhibiting the proliferation, invasion and metastasis of the cancer cells.
Zhonghua bing li xue za zhi Chinese journal of pathology 02/2010; 39(2):88-94.
-
[show abstract]
[hide abstract]
ABSTRACT: To study the complication incidence of 54 patients with chronic HBV infection following their orthotopic liver transplantation (OLT), and factors associated with HBV recurrence and hepatocellular carcinoma (HCC) recurrence or metastasis post-OLT.
The light-microscopic appearance of hepatic allograft biopsies in 54 patients with chronic HBV infection following OLT was examined. The related clinical data were analyzed. The incidence and occurrence time of post-OLT complications were studied. Furthermore, the relationship between recurrent type B viral hepatitis and acute rejection and the relationship among HCC recurrence/metastasis, acute rejection, the tumor diameter, and the portal vein invasion were particularly studied.
The frequent complications of patients with chronic HBV infection following OLT were acute rejection [38(70.4%); occurrence time: 5-365 d], chronic rejection [ 1(1.9%); occurrence time: 10.7 months],bile duct complications [24(44.4%); occurrence time: 7-940 d], HBV recurrence [7(13.0%); occurrence time: 1-540 d], HCV infection [3(5.6%); occurrence time: 60 d, 60 d, 33 months], CMV infection [8(14.8%); occurrence time: 67-90 d], and HCC recurrence or metastasis [17(31.5%); occurrence time: 2-41 months]. At the end of 1 year post-OLT, 95% of patients with post-hepatitis B cirrhosis were alive; At the end of 3 years post-OLT, 85% of patients with post-hepatitis B cirrhosis were alive. However, at the end of 1 year post-OLT, 67.6% of patients with post-hepatitis B HCC were alive; At the end of 3 years post-OLT, 50% of patients with post-hepatitis B HCC were alive. The number of acute rejection episodes in patients with recurrent HBV infection and that without recurrent HBV infection was (0.86+/-1.46) time/patient and (1.07+/-0.90) time/patient respectively(P>0.05); the number of moderate acute rejection episodes(RAI>or=score 4) in patients with recurrent HBV infection and that without recurrent HBV infection was (0.29+/-0.49) time/patient and (0.50+/-0.63) time/patient(P>0.05); Incidence of patients with >or=3 episodes of acute rejection in patient with recurrent HBV infection and that without recurrent HBV infection was 14.3% and 10.6%(P>0.05). Furthermore, the number of acute rejection episodes in patients with HCC recurrence or metastasis and that without HCC recurrence or metastasis was (1.12+/-0.93) time/patient and (1.06+/-1.39) time/patient respectively(P>0.05); the number of moderate acute rejection episodes(RAI>or=score 4) in patients with HCC recurrence or metastasis and that without HCC recurrence or metastasis was (0.65+/-0.79) time/patient and (0.65+/-1.06) time/patient respectively(P>0.05); Incidence of patients with >or=3 episodes of acute rejection in patient with HCC recurrence or metastasis and that without HCC recurrence or metastasis was 5.9% and 17.6% respectively(P>0.05). The tumor diameter in patients with HCC recurrence or metastasis was (6.72+/-3.40)cm, however, that in patients without HCC recurrence or metastasis was (3.55+/-2.17)cm(P=0.004 7).The incidence of Portal vein invasion in patients with HCC recurrence or metastasis and that without HCC recurrence or metastasis was 68.75% and 33.3% respectively(P=0.006).
There was no significant difference among HBV recurrence and acute rejection post-liver transplantation in patients with chronic HBV infection. There was no significant difference between HCC recurrence and acute rejection. The tumor diameter of patients with HCC recurrence or metastasis was significantly greater than that of no HCC recurrence or metastasis. The portal vein invasion of patients with HCC recurrence or metastasis was significantly frequent than that of no HCC recurrence or metastasis.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 08/2008; 40(4):408-14.
-
[show abstract]
[hide abstract]
ABSTRACT: To study the histopathologic features, differential diagnosis and prognosis of epithelioid angiomyolipoma (EAML) of kidney.
Two cases of EAML (including one case with recurrence) of kidney were retrieved from the archival files of Departments of Pathology, Navy General Hospital of PLA and Health Science Center of Peking University. The clinicopathologic features, immunohistochemistry, ultrastructural findings and follow-up data were studied and literature reviewed.
Histologically, the tumors were predominantly composed of epithelioid cells with marked cellular pleomorphism. Focal perivascular arrangement was seen. Hemorrhage and necrosis were present and tumor emboli were found in the venous structures. The renal hilar lymph nodes were also involved by tumor cells. Immunohistochemical study showed that the tumor cells (including those in the hilar lymph nodes) were strongly and diffusely positive for HMB45, smooth muscle actin, neuron-specific enolase and vimentin. They were focally positive for S-100 protein, melan-pan and CD68. The staining for epithelial membrane antigen, AE1/3, CK7, CD117, muscle-specific actin, desmin, leukocyte common antigen, CD20, CD45RO, CD30, CD15, chromogranin A, synaptophysin, bcl-2, estrogen receptor, progesterone receptor and p53 were negative. Ultrastructural examination revealed the presence of melanosome-like dense granules, myofilaments and dense bodies in the tumor cell cytoplasm. Discontinuous and focally thickened basal lamina was seen surrounding the tumor cells. On follow up, both patients remained well and disease-free 10 months after operation.
EAML is predominantly or almost entirely composed of epithelioid cells. Perivascular cellular arrangement, focal features of otherwise classic angiomyolipoma, as well as coexpression of HMB-45 and smooth muscle actin are clues to the correct diagnosis. The degree of cytologic atypia, presence of hemorrhage and necrosis and high mitotic activity may indicate the malignant potential of this tumor. On the other hand, the presence of lymph node involvement and even tumor emboli in renal veins may represent multifocaltumorigenicity rather than true malignancy. Definitive evidence of malignancy requires demonstration of distant metastasis.
Zhonghua bing li xue za zhi Chinese journal of pathology 02/2007; 36(1):19-23.
-
[show abstract]
[hide abstract]
ABSTRACT: To compare the advantages and disadvantages of three staining methods of HBV.
Normal Liver tissue and HBV-infected, HCV-infected, or dually infected (HBV and HCV) liver tissues were selected for this study. Formalin-fixed, paraffin-embedded sections (4 microm) were prepared. Each of the liver tissue specimens was detected by three staining methods, including immunohistochemical methods, Shikata's orcein stain and Victoria blue stain,respectively.
In the three methods, all of six HBV-infected cases showed intense staining, and three cases with dual infection (HBV and HCV) were weakly positive. However, both normal and HCV-infected liver tissues showed no staining. HBsAg stained dark brown with Immunohistochemical stain; HBsAg containing ground-glass hepatocytes stained magenta with Shikata's orcein stain; HBsAg stained blue with Victoria blue.
Each of three methods has its own advantages and disadvantages: high specificity and sensitivity, but high cost for immunohistochemical methods; complicated and overelaborate procedure for preparation of solutions, lower specificity and sensitivity, but low cost, for special staining methods.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 07/2006; 38(3):318-20.
-
Fei Pei,
Jie Zheng,
Zi-fen Gao,
Yan-feng Zhong,
Wei-gang Fang,
En-cong Gong,
Wan-zhong Zou,
Sheng-lan Wang,
Dong-xia Gao,
Zhi-gang Xie,
Min Lu,
Xue-ying Shi,
Cong-rong Liu,
Jing-ping Yang,
Yu-ping Wang,
Zhi-hui Han,
Xiao-hong Shi,
Wen-bin Dao,
Jiang Gu
[show abstract]
[hide abstract]
ABSTRACT: Severe acute respiratory syndrome (SARS) is an emerging infectious disease that first manifested in humans in November 2002. The SARS-associated coronavirus (SARS-CoV) has been identified as the causal agent, but the pathology and pathogenesis are still not quite clear.
Post-mortem lung samples from six patients who died from SARS from April to July 2003 were studied by light and electron microscopy, Masson trichromal staining and immunohistochemistry. Evidence of infection with the SARS-CoV was determined by reverse-transcription PCR (RT-PCR) , serological examination and electron microscopy.
Four of six patients had serological and RT-PCR evidence of recent infection of SARS-CoV. Morphologic changes are summarized as follows: (1) Diffuse and bilateral lung consolidation was seen in all patients (6/6) with increasing lung weight. (2) Diffuse alveolar damage was universal (6/6) with hyaline membrane formation (6/6), intra-alveolar edema/hemorrhage (6/6), fibrin deposition (6/6), pneumocyte desquamation (6/6). A marked disruption in the integrity of the alveolar epithelium was confirmed by immunostaining for the epithelial marker AE1/AE3 (6/6). (3) Type II pneumocytes, with mild hyperplasia, atypia, cytomegaly with granular amphophilic cytoplasm and intracytoplasmic lipid accumulation (5/6). (4) Giant cells in the alveoli were seen in five of 6 patients (5/6) , most of which were positive for the epithelial marker AE1/AE3 (5/6), but some cells were positive for the macrophage marker CD68(2/6). (5) A pronounced increase of macrophages were seen in the alveoli and the interstitium of the lung (6/6), which was confirmed by histological study and immunohistochemistry. (6) Haemophagocytosis was present in five of the 6 patients(5/6). (7) Lung fibrosis was seen in five patients(5/6), with alveolar septa and interstitium thickening(5/6), intraalveolar organizing exudates (6/6) and pleura thickening (4/6). Proliferation of collagen was confirmed by Masson trichromal staining, most of which was type III collagen by immunostaining. The formation of distinctive fibroblast/myofibroblast foci was seen in five patients (5/6) by light microscopy and immunochemistry. (8) Squamous metaplasia of bronchial mucosa was seen in five patients(5/6). (9) Thrombi was seen in all patients(6/6). (10) Accompanying infection was present in two patients, one was bacteria, the other was fungus. In addition, electron microscopy revealed viral particles in the cytoplasm of alveolar epithelial cells and endothelial cells corresponding to coronavirus.
Direct injury of SARS-CoV on alveolar epithelium, prominent macrophage infiltration and distinctive fibroblast/myofibroblast proliferation may play major roles in the pathogenesis of SARS.
Zhonghua bing li xue za zhi Chinese journal of pathology 11/2005; 34(10):656-60.
-
Jiang Gu,
Encong Gong,
Bo Zhang,
Jie Zheng,
Zifen Gao,
Yanfeng Zhong,
Wanzhong Zou,
Jun Zhan,
Shenglan Wang,
Zhigang Xie, [......],
Hongquan Shao,
Weigang Fang,
Dongshia Gao, Fei Pei,
Xingwang Li,
Zhongpin He,
Danzhen Xu,
Xeying Shi,
Virginia M Anderson,
Anthony S-Y Leong
[show abstract]
[hide abstract]
ABSTRACT: After >8,000 infections and >700 deaths worldwide, the pathogenesis of the new infectious disease, severe acute respiratory syndrome (SARS), remains poorly understood. We investigated 18 autopsies of patients who had suspected SARS; 8 cases were confirmed as SARS. We evaluated white blood cells from 22 confirmed SARS patients at various stages of the disease. T lymphocyte counts in 65 confirmed and 35 misdiagnosed SARS cases also were analyzed retrospectively. SARS viral particles and genomic sequence were detected in a large number of circulating lymphocytes, monocytes, and lymphoid tissues, as well as in the epithelial cells of the respiratory tract, the mucosa of the intestine, the epithelium of the renal distal tubules, the neurons of the brain, and macrophages in different organs. SARS virus seemed to be capable of infecting multiple cell types in several organs; immune cells and pulmonary epithelium were identified as the main sites of injury. A comprehensive theory of pathogenesis is proposed for SARS with immune and lung damage as key features.
Journal of Experimental Medicine 08/2005; 202(3):415-24. · 13.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To look for a rapid low-cost technique for the detection of HBV variants.
Two patients who underwent orthotopic liver transplantation (OLT) for HBV infection were treated with lamivudine (100 mg daily) and HBV infection recurred in the grafted livers. The patients were monitored intensively for liver enzymes, hepatitis B surface antigen (HBsAg) and HBV DNA in serum. Liver biopsy was performed regularly. HBV DNA in a conserved polymerase domain (the YMDD locus) was amplified from serum of each patient by PCR and sequenced. HBV genotypes were analyzed by restriction fragment length polymorphism (RFLP) of the PCR products generated from a fragment of the polymerase gene.
YMDD wild-type HBV was detected in one patient by PCR-RFLP and DNA sequencing 19 mo after OLT, and YIDD mutant-type HBV in the other patient, 16 mo after OLT.
PCR-RFLP assay is an accurate and simple method for genotyping lamivudine-resistant HBV variants.
World Journal of Gastroenterology 06/2005; 11(18):2714-9. · 2.47 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors.
By using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fusion protein was used to immunize BALB/c mice. The monoclonal antibody against G3BP was produced through conventional hybridoma method and characterized by ELISA, Western blot and immunohistochemical staining.
A hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the antibody was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively with the presence of lymph node metastasis and c-erbB2 expression.
The G3BP-specific monoclonal antibody derived from recombination protein can be used in ELISA, Western blot and immunohistochemical assay. It may provide an important tool in analysis of G3BP in in vitro and in vivo experiments. Besides, G3BP may serve as another prognostic marker for breast cancer.
Zhonghua bing li xue za zhi Chinese journal of pathology 05/2005; 34(4):215-9.
-
Xueying Shi,
Encong Gong,
Dongxia Gao,
Bo Zhang,
Jie Zheng,
Zifen Gao,
Yanfeng Zhong,
Wanzhong Zou,
Bingquan Wu,
Weigang Fang, [......],
Congrong Liu, Fei Pei,
Jingping Yang,
Yuping Wang,
Zhihui Han,
Xiaohong Shi,
Qianying Zhang,
Jiangfeng You,
Xiang Zhu,
Jiang Gu
[show abstract]
[hide abstract]
ABSTRACT: A significant percentage of confirmed severe acute respiratory syndrome (SARS) patients experienced gastrointestinal symptoms, and the viral sequence was detectable in the stool of most patients. At present, the knowledge of the pathology of the digestive system in SARS patients is limited. Because a resurgence of the SARS epidemic is constantly possible, there is an urgent need to understand the involvement of the digestive system in this new disease.
We performed seven SARS autopsies in which samples of alimentary tract and digestive glands were examined with routine pathology, electron microscopy (EM), in situ hybridization (ISH), immunohistochemistry, and real-time polymerase chain reaction (PCR).
The main histopathological finding was atrophy of the mucosal lymphoid tissue. A few mucosal epithelial cells and lymphocytes in the intestine were positively stained for coronavirus with ISH. SARS-coronavirus (CoV)-like particles were found in the mucosal epithelial cells under EM and mild focal inflammation was detected in the alimentary tract. One patient who experienced severe diarrhea had pseudomembranous enteritis of the ileum. Fatty degeneration and central lobular necrosis were observed in the liver. No evidence of direct viral infection was found in the esophagus, the stomach, the salivary gland, the liver, or the pancreas.
In addition to the lungs, the gastrointestinal tract is another target of SARS-CoV infection, as the intestinal epithelial cells and mucosal lymphoid tissue are infected. The findings provide possible explanations for the gastrointestinal symptoms and the presence of virus in the stool of SARS patients.
The American Journal of Gastroenterology 02/2005; 100(1):169-76. · 7.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In order to clarify the exact molecular weight of tumor metastasis suppressor gene-1 (TMSG-1) protein and its cellular localization, a monoclonal antibody against TMSG-1 was prepared, characterized and applied to evaluate the metastatic potential of human tumors.
A dominant epitope-TMSG-1(15)-derived from TMSG-1 was synthesized based on Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen preparation was used to immunize BAL B/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, which were further characterized by western blotting and immunohistochemical staining.
One hybridoma cell line secreting anti-TMSG-1 antibody, designated as C8, was eventually established after primary ELISA screening, followed by rapid limited dilution procedure. It was confirmed that C8 was of IgM isotype. Result of competitive inhibition assay showed that the antibody was TMSG-1 specific. Using this antibody, an expected protein band of about 45,000 (relative molecular mass) was detected in the non-metastatic variants PC(3)-2B4 and PG-LH7 cells by Western blotting, but not in the isogenetic metastatic variants of PC3-1E8 and PG-BE1 cells. Immunohistochemistry using C8 showed a positive staining of cell membrane and cytoplasm of 2B4 and LH7 cells, whereas 1E8 and BE1 cells were non-reactive. Immunostaining using C8 of paraffin sections of 52 breast carcinomas and 41 colon cancers demonstrated a strong positivity in non-metastatic tumors, but none to weakly reactive in metastatic tumors (P < 0.05).
C8 monoclonal antibody against the synthetic peptide is TMSG-1 specific and is effective for Western blot and immunohistochemistry assays to detect TMSG-1 expression in cancer cells. TMSG-1 protein is about 45 000 (relative molecular mass) at cell membrane and cytoplasm of tumor cells. Expression of TMSG-1 protein correlates well, inversely with the tumor metastatic potential.
Zhonghua bing li xue za zhi Chinese journal of pathology 01/2005; 34(1):15-21.
