Control of apoptosis during angiogenesis by survivin expression in endothelial cells

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to approximately 16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor alpha/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.     

VE-cadherin expression and clustering maintain low levels of survivin in endothelial cells

Survivin is strongly expressed in embryonic organs and in tumor cells but is low or absent in differentiated normal tissues. Resting endothelium expresses low levels of survivin but can up-regulate its synthesis on activation to proliferate. The mechanisms responsible for survivin down-regulation in resting conditions are still unknown. We report here that confluence and vascular endothelial-cadherin (VE-cadherin) expression induce contact inhibition of cell growth and survivin down-regulation in the endothelium. Using beta-catenin null and positive isogenic endothelial cell lines we found that the effect requires beta-catenin expression and its association to VE-cadherin cytoplasmic tail. Furthermore, in allantois organ cultures, survivin expression is up-regulated in areas of growing vessels where VE-cadherin is partially dismantled from junctions or in VE-cadherin -/- specimens. Overall, these data indicate that VE-cadherin and beta-catenin may negatively regulate survivin synthesis in endothelial cells. Consistently, in epidermal and pancreatic cell lines or ovarian tumors, epithelial-cadherin (E-cadherin) and survivin expression is inversely related, suggesting a non-cell-specific role of cadherins in reducing survivin synthesis.     

Gene alterations in tumor-associated endothelial cells from endometrial cancer

The alterations in the gene expression profile of tumor-associated human endometrial endothelial cells (HEECs) may allow opportunities for developing new therapeutic approaches to inhibit angiogenesis in endometrial cancer. The aim of this study was to identify the different gene expression pattern between tumor-associated HEECs and normal HEECs. To elucidate the molecular mechanisms governing the abnormal vasculature in endometrial cancer, we examined global expression patterns of purified endothelial cells from three endometrial cancers and three age-matched normal endometria using oligonucleotide microarrays. We also performed in vitro culture and identified the endothelial origin, as well as observing the functional characteristics in angiogenesis, of HEECs from the two different sources. Microarray analyses revealed distinct gene expression patterns and consistent up-regulation of certain endometrial endothelial marker genes across patient samples. More than 300 genes that exhibited > or =2-fold differences were identified in tumor-associated HEECs. Pathway analysis showed that pathways of Cell cycle, Cell adhesion molecules (CAMs), focal adhesion, and extracellular matrix (ECM)-receptor interaction were obviously predominant. The results of the microarray analysis were confirmed by quantitative real-time PCR, immunohistochemistry, and/or Western blotting. Moreover, although the tumor-associated HEECs did not show faster proliferation than normal HEECs, they exhibited enhanced migration ability, potent invasiveness, and elevated tube formation in vitro. The present study shows that tumor and normal endothelium differ at the molecular level, and additional characterization of this gene expression database will provide insights into the angiogenesis of endometrial cancers and might be of great benefit for finding potential therapeutic targets.     

构建survivin shRNA重组载体及靶向下调PC3细胞survivin mRNA的表达

背景：survivin基因特异性表达于肿瘤和胚胎组织,与肿瘤细胞的分化增殖、浸润转移以及多药耐药密切相关。 目的：构建靶向survivin基因的shRNA重组质粒表达载体,转染前列腺癌细胞PC3,验证该载体能否下调细胞survivin基因mRNA水平。 方法：以survivin基因为靶点设计具有短发夹结构的shRNA序列,经退火成互补双链后克隆入pENTR/U6建立重组表达载体pENTR/U6-SUR;转化E.coli TOP10菌株,挑取阳性菌落进行菌落PCR和测序鉴定;将重组质粒转染前列腺癌细胞株PC3细胞,RT-PCR检测重组质粒对细胞survivin基因mRNA水平的抑制效果。 结果与结论：将设计合成的shRNA序列经退火后克隆至pENTR/U6载体中,菌落PCR可扩增出目的条带,测序结果证实插入片段为所需序列;pENTR/U6-SUR重组质粒转染后PC3细胞survivin基因mRNA表达水平显著下降,且24 h比48 h作用更明显。成功构建了靶向survivin基因的shRNA质粒表达载体,并证实该载体显著下调了PC3细胞中survivin基因mRNA水平。     

Increased expression of P-glycoprotein and doxorubicin chemoresistance of metastatic breast cancer is regulated by miR-298

MicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate the expression of a number of genes involved in cancer; therefore, they offer great diagnostic and therapeutic targets. We have developed doxorubicin-resistant and -sensitive metastatic human breast cancer cell lines (MDA-MB-231) to study the chemoresistant mechanisms regulated by miRNAs. We found that doxorubicin localized exclusively to the cytoplasm and was unable to reach the nuclei of resistant tumor cells because of the increased nuclear expression of MDR1/P-glycoprotein (P-gp). An miRNA array between doxorubicin-sensitive and -resistant breast cancer cells showed that reduced expression of miR-298 in doxorubicin-resistant human breast cancer cells was associated with increased expression of P-gp. In a transient transfection experiment, miR-298 directly bound to the MDR1 3' untranslated region and regulated the expression of firefly luciferase reporter in a dose-dependent manner. Overexpression of miR-298 down-regulated P-gp expression, increasing nuclear accumulation of doxorubicin and cytotoxicity in doxorubicin-resistant breast cancer cells. Furthermore, down-regulation of miR-298 increased P-gp expression and induced doxorubicin resistance in sensitive breast cancer cells. In summary, these results suggest that miR-298 directly modulates P-gp expression and is associated with the chemoresistant mechanisms of metastatic human breast cancer. Therefore, miR-298 has diagnostic and therapeutic potential for predicting doxorubicin chemoresistance in human breast cancer.     

Cartilage oligometric matrix protein-angiopoietin-1 promotes revascularization through increased survivin expression in dermal endothelial cells of skin grafts in mice

The present study examined the effects of cartilage oligometric matrix protein angiopoietin-1 (COMP-Ang1) on the revascularization of mice skin grafts. Full-thickness skin grafts were autotransferred into BALB/c mice. The donor grafts were soaked in COMP-Ang1 protein (50 mug/ml, n = 10) or in bovine serum albumin (BSA) (50 mug/ml, n = 10) dissolved in 1 ml of sterile, phosphate-buffered saline for 5 minutes before transfer. Revascularization of the grafts was monitored using an intravital microscope on postoperative days 3, 4, and 5. Morphological and immunohistochemical analyses were performed to evaluate platelet-endothelial cell adhesion molecule-1 and survivin expression and apoptotic signal in the transplanted grafts. Grafts soaked in COMP-Ang1 (COMP-Ang1 group) showed significantly increased revascularization compared with grafts soaked in BSA (BSA group) on intravital microscopy and platelet-endothelial cell adhesion molecule-1 staining. The COMP-Ang1 group showed a significant increase of survivin expression in the endothelial cells and a reduction of apoptotic signal in comparison to the BSA group. Therefore, we believe that COMP-Ang1 provides the therapeutic benefit of enhancing the survival of vascular endothelial cells during transplantation of skin graft.     

Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin

Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.     

siRNA抑制胃癌细胞BGC-823及裸鼠皮下移植瘤中生存素的表达

目的： 探讨RNA干扰用于胃癌治疗的可行性与特异性。方法: 设计靶向生存素基因的小干扰RNA,并导入胃癌BGC-823细胞株和裸鼠皮下移植瘤,在体内、外诱导RNA干扰,采用MTT、流式细胞检测技术、RT-PCR法、Western bloting、免疫组织化学和TUNEL检测survivin基因表达、细胞周期和细胞凋亡。结果: 重组质粒pTZU6+1-siRNA- survivin导入BGC-823后,survivin mRNA和蛋白表达均明显下调;细胞生长被抑制,细胞周期中S期细胞减少, G1/G0期细胞增加,出现明显细胞凋亡。裸鼠皮下移植瘤体积明显缩小,Survivin表达均下调。体内、外对照组各指标均无明显变化。 结论: RNA干扰明显抑制靶基因survivin在胃癌细胞BGC-823及裸鼠皮下移植瘤中的表达及肿瘤细胞增殖,并诱导细胞凋亡,且特异性好,可能成为肿瘤治疗的新方法。     

The surface expression of a tumor-associated antigen on human kappa myeloma cells

The monoclonal antibody K-1-21 defines an antigen, KMA (kappa myeloma antigen) on the surface of human kappa myeloma cells. K-1-21 also recognizes human kappa light chains in free form but not when covalently bonded to heavy chains. To examine the relationship between KMA and this determinant on free kappa chains, the surface expression of KMA was examined on the IgG, kappa myeloma line LICR LON/HMy2 (HMy2). No patching or capping was observed in the presence of K-1-21 alone, but KMA could be capped if the cells were incubated with K-1-21 followed by fluorescein isothiocyanate-conjugated sheep F(ab')2 anti-mouse immunoglobulin. Capping was not affected by the inhibitors calcium ionophore or dibucaine. When IgG molecules were removed from the cell surface by capping with anti-IgG antiserum both KMA and free kappa light chains could still be detected with K-1-21 and a polyvalent anti-kappa antiserum, respectively. By contrast, after removal of all surface kappa chains with the polyvalent anti-kappa serum, no staining was observed with K-1-21 indicating that KMA may be an epitope on free kappa chains inserted in the membrane of kappa myeloma cells but absent from normal cells. KMA cell surface expression varied with the stage of the cell cycle. Flow cytometric analysis of K-1-21-stained HMy2 cells from either continuous cultures or from elutriated fractions enriched for various cell cycle phases showed that, within the cycling population, cells in G2 + M expressed KMA at a higher frequency and density than did cells in G1.     

Calprotectin expression in vitro by oral epithelial cells confers resistance to infection by Porphyromonas gingivalis

Calprotectin, an S100 calcium-binding protein with broad-spectrum antimicrobial activity in vitro, is expressed in neutrophils, monocytes, and gingival keratinocytes. In periodontitis, calprotectin appears upregulated and is detected at higher levels in gingival crevicular fluid and tissue specimens. How calprotectin contributes to the pathogenesis of periodontal diseases is unknown. To isolate the effects of calprotectin, a calprotectin-negative oral epithelial cell line was transfected with calprotectin genes to enable expression. Porphyromonas gingivalis was permitted to bind and invade transfected cells expressing calprotectin and sham transfectants. Rates of invasion into both cell lines were compared using the antibiotic protection assay. Transfected cells expressing calprotectin showed 40 to 50% fewer internalized P. gingivalis than sham transfectants. Similarly, binding to calprotectin expressing cells was reduced approximately twofold at all time points (15, 30, 45, and 60 min) as estimated by immunofluorescence analysis. Independent of invasion, however, prolonged exposure to P. gingivalis induced epithelial cell rounding and detachment from the substratum. These morphological changes were delayed, however, in cells expressing calprotectin. Using P. gingivalis protease-deficient mutants, we found that Arg-gingipain and Lys-gingipain contributed to epithelial cell rounding and detachment. In conclusion, expression of calprotectin appears to protect epithelial cells in culture against binding and invasion by P. gingivalis. In addition, cells expressing calprotectin are more resistant to detachment mediated by Arg-gingipain and Lys-gingipain. In periodontal disease, calprotectin may augment both the barrier protection and innate immune functions of the gingival epithelium to promote resistance to P. gingivalis infection.     

Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in the metastatic 4T1 breast cancer model

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.     

Go and chemoresistance of malignant cells

In general terms the paper discusses the relevance of mitotically quiescent, malignant cells for increasing resistance to chemotherapy. Detailed are some routes to Go for malignancies either dependent or autonomous for their mitotic potential.     

Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting

The protective genes that mediate endothelial cell (EC) survival during angiogenesis have not been completely characterized. Here, we show that an antisense oligonucleotide to the apoptosis inhibitor survivin suppressed de novo expression of survivin in ECs by vascular endothelial cell growth factor (VEGF). In contrast, the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2 levels in endothelium. When assessed in cell death assays, antisense targeting of survivin abolished the anti-apoptotic function of VEGF against tumor necrosis factor-alpha- or ceramide-induced cell death, enhanced caspase-3 activity, promoted the generation of a approximately 17-kd active caspase-3 subunit, and increased cleavage of the caspase substrate, polyADP ribose polymerase. In contrast, the survivin antisense oligonucleotide had no effect on EC viability in the absence of VEGF. Antisense oligonucleotides to platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), lymphocyte function-associated molecule-3 (LFA-3, CD58), or intercellular adhesion molecule-1 (ICAM-1, CD54) did not reduce the anti-apoptotic function of VEGF in endothelium. When tested on other angiogenic activities mediated by VEGF, survivin antisense treatment induced rapid regression of three-dimensional vascular capillary networks, but did not affect EC migration/chemotaxis. These data suggest that the anti-apoptotic properties of VEGF during angiogenesis are primarily mediated by the induced expression of survivin in ECS: Manipulation of this pathway may increase EC viability in compensatory angiogenesis or facilitate EC apoptosis and promote vascular regression during tumor angiogenesis.     

Blockade of epidermal growth factor receptor signaling on tumor cells and tumor-associated endothelial cells for therapy of human carcinomas

The purpose of this study was to determine whether the expression of epidermal growth factor receptor (EGF-R) and activated EGF-R by tumor-associated endothelial cells is influenced by interaction with specific growth factors in the microenvironment. Different human carcinoma cell lines expressing EGF-R with low or high levels of EGF/transforming growth factor (TGF)-alpha were implanted into orthotopic organs of nude mice. In the EGF/TGF-alpha-positive bladder cancer (253J-BV), pancreatic cancer (L3.6pl), and renal cancer (RBM1-IT) but not in the EGF/TGF-alpha-negative renal cancer SN12-PM6, tumor-associated endothelial cells expressed EGF-R and activated EGF-R. Mice were implanted with human 253J-BV bladder tumors (EGF+) or human SN12-PM6 renal tumors (EGF-). Treatment with oral PKI 166 (a specific inhibitor of EGF-R phosphorylation) alone, intraperitoneal paclitaxel alone (253J-BV), gemcitabine alone (SN12-PM6), or combination of PKI 166 and chemotherapy produced a 60%, 32%, or 81% reduction in the volume of 253J-BV bladder tumors, respectively, and 26%, 23%, or 51% reduction in the volume of SN12-PM6 kidney tumors, respectively. Immunohistochemical analyses demonstrated down-regulation of activated EGF-R in EGF/TGF-alpha-positive and EGF/TGF-alpha-negative lesions from mice treated with PKI 166, although apoptosis of tumor-associated endothelial cells was found only in EGF/TGF-alpha-positive tumors. Collectively, these data suggest that expression of activated EGF-R by tumor-associated endothelial cells provides an important target for therapy.     

Expression of endothelial adhesion molecules in vivo. Increased endothelial ICAM-2 expression in lymphoid malignancies.

The authors have compared the reactivity of monoclonal antibodies (MAb) directed against endothelial adhesion molecules (ICAM-1, ICAM-2, VCAM-1, ELAM-1) in hyperplastic, nonmalignant, and malignant lymph nodes. The authors demonstrate that the reactivity with ICAM-1 MAb is stronger in the high endothelial venules (HEV) and other smaller vessels (SV) in lymphomas compared with hyperplastic lymph nodes. Similarly, the reactivity of an ICAM-2 MAb (6D5) was shown to be higher in malignant lymph nodes compared with nonmalignant ones. ICAM-2 MAb stained both the HEV and SV. VCAM-1 MAb reacted strongly with germinal centers and its endothelial reactivity was higher in the lymphoma nodes. ELAM-1 MAb stained only faintly some endothelial cells in malignant tissue. These data suggest that besides the known regulatable endothelial adhesion molecules ICAM-1 and VCAM-1, the expression of ICAM-2 can be modified.     

Increased glucocorticoid receptor Beta expression converts mouse hybridoma cells to a corticosteroid-insensitive phenotype

Glucocorticoid (GC) insensitivity is a challenging clinical problem associated with many chronic inflammatory disorders and life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (GCR) pre-mRNA generates a second GCR, termed GCRbeta, which does not bind GC but antagonizes the transactivating activity of the classic GCR, termed GCRalpha. GC-insensitive conditions have been associated with increased GCRbeta expression. Whether or not increased GCRbeta expression can contribute to GC insensitivity, however, remains controversial. To more precisely demonstrate the effect of GCRbeta on steroid responsiveness, we virally transduced GCRbeta cDNA into mouse DO-11.10 hybridoma cells, as mice are known to be deficient in the GCRbeta gene. We demonstrate that viral transduction of GCRbeta cDNA into mouse hybridoma cells to induce stable expression of GCRbeta results in GC insensitivity of these cells. Furthermore, in such cells GCRalpha is complexed with GCRbeta. Such heterodimer formation may account for the reduced effectiveness of GC action in cells overexpressing GCRbeta.