Histologic classification and prognosis factors in phyllodes tumors of breast / 中华病理学杂志

Objective@#To study the relationship between morphological characteristics, grading, diagnosis and prognosis in phyllodes tumors (PT) of the breast.@*Methods@#A retrospective study was carried out on 83 PTs diagnosed between 1999 and 2003 that were classified semi-quantitatively according to the WHO recommendation. Follow-up data was available for some cases, and Cox regression analysis was used to evaluate factors affecting metastasis and recurrence.@*Results@#All cases were classified into the benign (57.8%), borderline (28.9%) and malignant (13.3%). The overall recurrence rate for the 72 cases with follow-up data was 20.8% (15/72), and was 17.5% (7/40) in benign, 22.7% (5/22) in borderline and 3/10 in malignant PT, respectively, with no significant difference (P>0.05). The median interval between the initial diagnosis and the first recurrence was 24 months. Lung or bone metastases occurred in 1/22 borderline and 3/10 malignant PT patients 5 years post-surgery. The mitotic count and the degree of stromal cell atypia were significantly correlated with recurrence (P=0.001 and P=0.006). Multivariate analysis showed that severe stromal cell atypia was an independent predictor of recurrence-free survival in PT [HR=6.40 (95% CI=1.378 to 29.732), P=0.018].@*Conclusions@#Each parameter in the histological grading of PT may have different prognostic value, and markedly increased mitotic count and were predictive of relapse.

Construction of human Ewing sarcoma cell line with knockdown of inhibitor of differentiation 2 gene / 肿瘤研究与临床

Objective To construct short hairpin RNA (shRNA) expression plasmids against the inhibitor of differentiation 2 (Id2) gene and establish a suitable cell model for the role of Id2 in proliferation and differentiation of human Ewing sarcoma cell.Methods Three shRNA sequences targeting Id2 gene were designed and inserted into the pGPU6/GFP/Neo (-shRNA-Id2) expression vectors.The recombinant pGPU6/GFP/Neo-shRNA-Id2 plasmids were introduced into Ewing sarcoma RD-ES cells by liposome-mediated transfection.The knock-down efficiency of Id2 in infected RD-ES cells was verified by Western blot assay.The cell growth and cell cycle changes were evaluated by cell counting and flow cytometry between transfected cells and control cells.Results The Id2 expression decreased 54 ％ and 57 ％,respectively,in RD-ES cell line which were transfected with the shRNA-Id2-543 and shRNA-Id2-593 plasmids compared with the control group cells by Western blot analysis.The cell growth assay demonstrated that the cell number in transfected cells was significantly decreased during 6-7 d compared with the control group (P ＜ 0.05).The cells at the S-phase of cell cycle were increased [(36.60±1.53) ％ and (44.89E2.46) ％ vs (29.73±2.03) ％,P ＜ 0.05],and no significant changes at the G2 phase or even reduction in the transfected cells.Conclusions Id2 stable knock-down cell lines are successfully established.The reduced expression of Id2 is related with decreased cell growth and cell cycle arrest in the S-phase.Id2 maybe plays an important role in proliferation of Ewing sarcoma cell.

Fluorescence in-situ hybridization as a diagnostic tool for cutaneous melanoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the utility of fluorescence in situ hybridization as a diagnostic tool for cutaneous melanoma.</p><p><b>METHODS</b>Twenty cutaneous melanomas and 20 cutaneous nevi from pathology files were selected and analyzed by Vysis melanoma FISH probe kit targeting 3 loci on chromosome 6 (MYB, CEP6 and RREB1) and 1 locus on 11q (CCND1) and data were interpreted based on the Abbott criteria provided by the kit.</p><p><b>RESULTS</b>Informative FISH results were obtained in 16 melanomas and 18 nevi. Chromosomal aberrations were detected in 12 of the 16 melanomas and only 1 of 18 nevi.</p><p><b>CONCLUSION</b>FISH is a useful diagnostic tool and able to distinguish cutaneous nevus from melanoma with good sensitivity and specificity.</p>

LASS2/TMSG1 gene silencing promotes the invasiveness and metastatic of human prostatic carcinoma cells through increase in vacuolar ATPase activity / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the effects of LASS2/TMSG1 silencing on the growth, invasion and metastasis of prostate carcinoma cells and to investigate the related molecular mechanisms.</p><p><b>METHODS</b>LASS2/TMSG1 expression of human prostate carcinoma cell line with low metastatic potentiality (PC-3M-2B4 cells) was knocked down using DNA vector-based small interfering RNA (shRNA), followed by evaluations of tumor cell invasion and metastasis.</p><p><b>RESULTS</b>A stable PC-3M-2B4 cell line with expression of LASS2/TMSG1-shRNA was successfully established. MTT assay showed PC-3M-2B4 cells exhibited a strong proliferation after transfection of LASS2/TMSG1-shRNA.LASS2/TMSG1-shRNA transfected clones demonstrated an increased clonogenicity by soft agar colony formation assay and a significant increase of tumor cell invasion by matrigel invasion study.Flow cytometry showed that after LASS2/TMSG1 gene silencing, the apoptotic rate of PC-3M-2B4 cell significantly decreased (P<0.01) without significant cell cycle change (P>0.05).Eight weeks after implantation into subcutaneous tissues in BAL B/c (nu+) mice, the size and weight of sh-LASS2/TMSG1 xenografts were significantly larger than those of the control group (P<0.05).Nuclear proliferation index of the subcutaneous tumor was also higher in the LASS2/TMSG1 shRNA group than those in the control group. Lymph node metastasis was observed in 5 of 6 mice of LASS2/TMSG1 shRNA group and only 1 of 6 of the control group. V-ATPase activity, activities of secreted MMP-2 and MMP-9 and extracellular hydrogen ion concentration were significantly increased in LASS2/TMSG1-shRNA group compared with the control group (P<0.05).</p><p><b>CONCLUSION</b>Silencing of LASS2/TMSG1 promotes the growth, invasion and metastasis of prostate cancer cells through up-regulation of V-ATPase activity, indicating that LASS2/TMSG1 is a tumor metastasis suppressor gene.</p>

Biological effects of connective tissue growth factor transfection on human breast cancer cells / 中国癌症杂志

Background and purpose:Connective tissue growth factor(CTGF) is a member of CCN family,it has been reported that CTGF involve in many biological processes and various pathological conditions.In our study,the correlation of CTGF expression and biological effects on breast cancer cell lines were investigated.Methods:The eukaryotic expression vectors containing CTGF open reading frame(ORF) pcDNA3.0/CTGF were constructed and transfected into breast cancer cell lines.The relationship between CTGF expression and breast cancer cell growth ability and proliferation,cell cycle distribution,apoptosis,cell motility and invasive capacity in vitro were observed.Results:Upregulation of CTGF expression in MCF-7 cell line could inhibit its growth ability and proliferation,increased the proportion of G0G1 phase cells,enhanced apoptosis and inhibited its invasive ability in vitro.Downregulation of CTGF expression in MDA-MB-231 cell line increased its growth ability and proliferation,decreased apoptosis and promoted its invasive ability in vitro.There were no differences of cell motility among different groups in MCF-7 and MDA-MB-231 cell lines.Conclusions:CTGF can inhibit breast cancer cell growth by increasing cell apoptosis and/or the proportion of cells in G0G1 phase.CTGF can also inhibit breast cancer cell invasive ability.

Monoclonal antibodies against human tumor metastasis suppressor gene-1 (TMSG-1): preparation, characterization, and application.

To better understand the molecular mechanism of metastasis, the monoclonal antibody against tumor metastasis suppressor gene-1 (TMSG-1, one novel gene) was prepared, characterized, and applied in estimating the metastatic potential of human tumors. A dominant epitope, TMSG-1(15)--derived from TMSG-1--was automatically synthesized based on the Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen solution was used to immunize BALB/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and their characterization was performed by Western blotting and immunohistochemical staining. The hybridoma cell line secreting anti-TMSG-1 antibody, designated C8, was established after primary ELISA screening and consequent rapid limited dilution. C8 was IgM in isotyping. The competitive inhibition assay showed that the antibody was TMSG-1 specific. Furthermore, in Western blotting, a protein band of about 45 kD was detected with nonmetastatic variant PC-3M-2B4 and PG-LH7 cells, but not with the isogenetic metastatic variant PC-3M-1E8 and PG-BE1 cells. Immunohistochemistry method showed that positive staining presented in the cytomembrane and cytoplasm of 2B4 and LH7 cells, while 1E8 and BE1 cells were non-reactive. The sections from paraffin-embedded blocks of human cancer (52 cases of breast carcinoma and 41 cases of colon cancer) were stained, showing strongly positive in non-metastatic tumor and weakly positive or negative in metastatic tumor; the strongly positive rate of TMSG-1 expression in human cancers were, respectively, 36%, 7.4%, 52.4%, and 35% in non-metastatic and metastatic breast cancer and non-metastatic and metastatic colon cancer (p = 0.02). The monoclonal antibody developed against synthetic peptide was TMSG-1 specific, and it has promise in Western blot and immunohistochemistry for detecting the TMSG-1 expression of cancer cells and tissues. It may provide an important tool in the study of TMSG-1 expression and function in both experimental and clinical studies. Furthermore, our tests confirmed that TMSG-1 protein has excellent inverse correlation to tumor metastasis potential, with the molecular weight of 45 kD (supporting the encoded protein containing 380 amino acids) and localization in cytomembrane and cytoplasm of tumor cell.

Axonal regulation of Schwann cell differentiation and integrin α6β4 expression / 北京大学学报(医学版)

Objective: To study the axonal effect and the expression of integrin α6β4 during Schwann cell(SC) differentiation and myelination. Methods: Schwann cells were dissociated from the sciatic nerve of neonatal Waster rats and neurons dissociated from spinal cord. Singal cultures and purified populations of SC were cocultured with NC. Four methods (contrast microscope, scanning electron microscopy(SEM), immunocytochemistry method and in situ hybridization ) were used. Results: The separately cultured Schwann cells showed MBP negetive by immunocytochemistry method. But cocultured SC were shown positive. SEM showed that Schwann cells' membrane loop progressively circumnavigated around the axon during myelination, which suggested that the non-myelinating SC(nMSC) transformed to myelinating SC (MSC). In situ hybridization showed integrin α6β4 positive signals only on the outer surface of the Schwann cell-axon unit in SC coculture with NC. Conclusion: The differentiation and maturation of SC depend on axon, and the activity of integrins is expressed by axon. Axonal contact induces the expression of α6β4 during SC myelination, which suggests that integrin α6β4 is an important mediator of interactions of myelinating SC with the basal limina.

Effect of huayu xiaoliu fang on cell cycle of a human lung carcinoma cell line / 中国病理生理杂志

AIM: To investigate the effect of huayu xiaoliu fang, a Chinese medicine, on the cell cycle of human lung carcinoma cell line by serologic pharmacological method. METHODS: PGLH7 cells were incubated with rabbit serum containing huayu xiaoliu fang at different doses obtained by serologic pharmacological method. MTT assay was used to calculate the proliferation inhibition rate. The target cells were harvested to analyze the cell cycles by flow cytometry. RESULTS: The Chinese medicine-containing serum inhibited the growth of PGLH7 cells significantly. There was remarkable difference in the proliferation inhibition rate between 10% (high dose) Chinese medicine-containing serum and the control serum (P