Response gene to complement 32 is required for C5b-9 induced cell cycle activation in endothelial cells

Proliferation of vascular endothelial cells (EC) and smooth muscle cells (SMC) is a critical event in angiogenesis and atherosclerosis. We previously showed that the C5b-9 assembly during complement activation induces cell cycle in human aortic EC (AEC) and SMC. C5b-9 can induce the expression of Response Gene to Complement (RGC)-32 and over expression of this gene leads to cell cycle activation. Therefore, the present study was carried out to test the requirement of endogenous RGC-32 for the cell cycle activation induced by C5b-9 by knocking-down its expression using siRNA. We identified two RGC-32 siRNAs that can markedly reduce the expression of RGC-32 mRNA in AEC. RGC-32 silencing in these cells abolished DNA synthesis induced by C5b-9 and serum growth factors, indicating the requirement of RGC-32 activity for S-phase entry. RGC-32 siRNA knockdown also significantly reduced the C5b-9 induced CDC2 activation and Akt phosphorylation. CDC2 does not play a role in G1/S transition in HeLa cells stably overexpressing RGC-32. RGC-32 was found to physically associate with Akt and was phosphorylated by Akt in vitro. Mutation of RGC-32 protein at Ser 45 and Ser 47 prevented Akt mediated phosphorylation. In addition, RGC-32 was found to regulate the release of growth factors from AEC. All these data together suggest that cell cycle induction by C5b-9 in AEC is RGC-32 dependent and this is in part through regulation of Akt and growth factor release.     

Epigenetic modifications induced by RGC-32 in colon cancer

First described as a cell cycle activator, RGC-32 is both an activator and a substrate for CDC2. Deregulation of RGC-32 expression has been detected in a wide variety of human cancers. We have now shown that RGC-32 is expressed in precancerous states, and its expression is significantly higher in adenomas than in normal colon tissue. The expression of RGC-32 was higher in advanced stages of colon cancer than in precancerous states or the initial stages of colon cancer. In order to identify the genes that are regulated by RGC-32, we used gene array analysis to investigate the effect of RGC-32 knockdown on gene expression in the SW480 colon cancer cell line. Of the 230 genes that were differentially regulated after RGC-32 knockdown, a group of genes involved in chromatin assembly were the most significantly regulated in these cells: RGC-32 knockdown induced an increase in acetylation of histones H2B lysine 5 (H2BK5), H2BK15, H3K9, H3K18, and H4K8. RGC-32 silencing was also associated with decreased expression of SIRT1 and decreased trimethylation of histone H3K27 (H3K27me3). In addition, RGC-32 knockdown caused a significantly higher percentage of SW480 cells to enter S phase and subsequently G2/M. These data suggest that RGC-32 may contribute to the development of colon cancer by regulating chromatin assembly.     

Rearrangements and increased expression of cyclin D1 (CCND1) in neuroblastoma

Cyclin D1 regulates G1 cell cycle progression by controlling the phosphorylation of the retinoblastoma protein. This pathway is frequently deregulated in many malignancies. In neuroblastoma, however, no consistent G1 cell cycle checkpoint aberrations have been found. We examined the possible deregulation of cyclin D1 (CCND1) in this tumor. mRNA expression profiles of neuroblastoma generated by SAGE (Serial Analysis of Gene Expression) revealed a high expression of CCND1 in a subset of neuroblastoma cell lines and tumors. The CCND1 expression level can be 0.3% of the total cellular mRNA. Northern blot analysis of CCND1 expression showed a relative overexpression in 16 of 23 neuroblastoma cell lines and 10 of 15 tumor samples. In the majority of cases, the high CCND1 mRNA levels also led to high CCND1 protein levels. In the search for mechanisms causing this relative overexpression, we screened for amplifications and rearrangements of CCND1. Five amplifications were found in 202 neuroblastoma tumors and cell lines. Analysis of the 3'-UTR of CCND1 showed a rearrangement in 1 of 96 tumors. These clonal aberrations of CCND1 together with the high expression suggest a role for deregulated CCND1 activity in neuroblastoma tumorigenesis.     

Expression and localization of GRP75 in human epithelial tumors and normal tissues.

Using differential display mRNA techniques, the authors found cDNA of the heat shock 70 protein known as GRP75 overexpressed in ovarian cancer cell lines. In the current study, the authors used immunohistochemistry to characterize the expression pattern of GRP75 in ovarian carcinomas and compared it with epithelial tumors originating from the female reproductive tract, epithelial neoplasms from non-gynecologic sites (colon, pancreas, breast, and lung), and various normal tissues. The authors also developed an antigen capture ELISA assay to determine if GRP75 can be detected in tumors, ascites, or sera of patients with advanced mullerian adenocarcinomas. All epithelial tumors from the ovary and the female reproductive tract were positive for GRP75 expression with moderate to strong staining intensity; stromal expression of GRP75 was generally weak or absent. Adenocarcinomas from the colon, lung, pancreas, and breast also stained strongly positive for GRP75. The epithelial cells of all normal tissues examined were positive for GRP75, and strong staining was also seen in the corpora lutea, hepatocytes, enteric neural plexus of the esophagus and colon, and placental cytotrophoblast and syncytiotrophoblast, and in subpopulations of pancreatic acinar cells. The ELISA assay detected GRP75 in tumor lysates and ascitic fluid, but not sera, of patients with mullerian adenocarcinomas. The authors conclude that GRP75 is highly expressed in both benign and malignant epithelium, as well as cells of specialized function from a variety of tissues.     

Progressive deregulation of the cell cycle with higher tumor grade in the stroma of breast phyllodes tumors

We studied cell cycle-regulating proteins in phyllodes tumor pathogenesis by immunohistochemical analysis for Ki-67, cyclin A, cyclin D1, retinoblastoma protein (pRb), p53, p16INK4A, bcl-2, and p21waf1 in the epithelium and stroma of 40 primary (benign, 21; borderline, 8; malignant, 11) and 7 recurrent tumors of different grades. In most cases, the epithelium showed no altered expression of cell cycle regulators. Stromal overexpression of p16INK4A, p53, cyclin A, pRb, and p21waf1 correlated significantly with tumor grade. The number of altered proteins in stroma increased with higher grade and was accompanied by increased proliferation. Stromal cyclin A expression was the best separating marker between tumor grades. Correlations existed between stromal overexpression of p16NK4A and p21waf1, p16INK4A and p53, and p53 and pRb. No immunostaining differences were detected between primary tumors and recurrences. Four or more altered proteins and p53 expression in the stromal component were independent negative prognosticators for disease-free survival. The stromal component of mammary phyllodes tumors displays an increasing level of cell cycle deregulation with higher tumor grade; the epithelial compartment mostly remains inconspicuous. Several combinations of aberrantly expressed cell cycle proteins seem important in the stromal progression of phyllodes tumors. The number of stromal cell cycle aberrations and stromal p53 expression might predict clinical behavior.     

Dual role of Response gene to complement-32 in multiple sclerosis

Response gene to complement (RGC)-32 is a novel molecule that plays an important role in cell proliferation. We investigated the expression of RGC-32 in multiple sclerosis (MS) brain and in peripheral blood mononuclear cells (PBMCs) obtained from patients with relapsing–remitting multiple sclerosis. We found that CD3⁺, CD68⁺, and glial fibrillar acidic protein (GFAP)⁺ cells in MS plaques co-localized with RGC-32. Our results show a statistically significant decrease in RGC-32 mRNA expression in PBMCs during relapses when compared to the levels in stable MS patients. This decrease might be useful in predicting disease activity in patients with relapsing–remitting MS. RGC-32 expression was also correlated with that of FasL mRNA during relapses. FasL mRNA expression was significantly reduced after RGC-32 silencing, indicating a role for RGC-32 in the regulation of FasL expression. In addition, the expression of Akt1, cyclin D1, and IL-21 mRNA was significantly increased during MS relapses when compared to levels in healthy controls. Furthermore, we investigated the role of RGC-32 in TGF-β-induced extracellular matrix expression in astrocytes. Blockage of RGC-32 using small interfering RNA significantly inhibits TGF-β induction of procollagen I, fibronectin and of the reactive astrocyte marker α-smooth muscle actin (α-SMA). Our data suggest that RGC-32 plays a dual role in MS, both as a regulator of T-cells mediated apoptosis and as a promoter of TGF-β-mediated profibrotic effects in astrocytes.     

Thymosin-beta4 regulates motility and metastasis of malignant mouse fibrosarcoma cells

We identified a thymosin-beta4 gene overexpression in malignant mouse fibrosarcoma cells (QRsP-30) that were derived from clonal weakly tumorigenic and nonmetastatic QR-32 cells by using a differential display method. Thymosin-beta4 is known as a 4.9-kd polypeptide that interacts with G-actin and functions as a major actin-sequestering protein in cells. All of the six malignant fibrosarcoma cell lines that have been independently converted from QR-32 cells expressed high levels of thymosin-beta4 mRNA and its expression in tumor cells was correlated with tumorigenicity and metastatic potential. Up-regulation of thymosin-beta4 in QR-32 cells (32-S) transfected with sense thymosin-beta4 cDNA converted the cells to develop tumors and formed numerous lung metastases in syngeneic C57BL/6 mice. In contrast, antisense thymosin-beta4 cDNA-transfected QRsP-30 (30-AS) cells reduced thymosin-beta4 expression, and significantly lost tumor formation and metastases to distant organs. Vector-alone transfected cells (32-V or 30-V cells) behaved like their parental cells. We observed that tumor cell motility, cell shape, and F-actin organization is regulated in proportion to the level of thymosin-beta4 expression. These findings indicate that thymosin-beta4 molecule regulates fibrosarcoma cell tumorigenicity and metastasis through actin-based cytoskeletal organization.     

Immune recognition of cyclin B1 as a tumor antigen is a result of its overexpression in human tumors that is caused by non-functional p53

Cyclin B1, which plays a key role in the control of cell cycle progression from G(2) through M phase, was recently identified by us as a tumor antigen recognized by human T-cells. To understand what makes this normal molecule antigenic, we compared its expression in malignant versus normal cells. Immunohistology showed overexpression of cyclin B1 protein in tumors compared to surrounding normal tissue and localization in the cytoplasm rather than the nucleus. Cyclin B1 is overexpressed at protein and mRNA level in many tumor cell lines including breast, lung, colorectal carcinoma, lymphoma and leukemia. While overexpressed in tumor cells at all stages of the cell cycle, its expression still peaks at G(2)/M phase, as it does in normal cells. We compared cyclin B1 expression in two cell clones derived from the same colorectal tumor cell line, one wild type for p53 (HCT116p53(+/+)) and one with deleted p53 (HCT116p53(-/-)). HCT116p53(+/+) cells had undetectable (normal) level of cyclin B1 protein, while HCT116p53(-/-) cells showed overexpression. When reconstituted with p53, HCT116p53(-/-) cells reverted to normal cyclin B1 expression. We conclude that p53 plays an important role in cyclin B1 regulation and that tumors with mutated p53 will be good candidates for cyclin B1 based immunotherapy.     

过表达miR-10b促进肺癌细胞系A549增殖

目的探讨非小细胞肺癌(NSCLC)患者肺癌及癌旁组织中miR-10b(miR-10b)表达水平及miR-10b是否通过调控锌指转录蛋白基因(KLF4)对肺癌细胞系A549恶性化的影响。方法 40例NSCLC患者病理切片,原位杂交检测肺癌及癌旁组织中miR-10b的表达量;对肺癌细胞系A549转染miR-10b mimics后,CCK-8法检测肺癌细胞增殖;real-time PCR及Western blot检测KLF4 mRNA及蛋白水平;软琼脂克隆形成实验检测过表达miR-10b对A549细胞的肿瘤恶性化程度的影响。结果肺癌细胞A549及肺癌组织中miR-10b的表达量分别高于正常肺上皮细胞16HBE及癌旁组织;过表达miR-10b模拟物的A549细胞中,KLF4蛋白水平显著下降(P0.05);过表达的miR-10b可显著增加A549细胞的增殖速度及在软琼脂内的成瘤性。结论 miR-10b在不同类型细胞及组织中具有分布差异性、可能是通过抑制KLF4的表达促进肺癌细胞增殖及恶性化。     

Expression of the ELAV-like protein HuR in human colon cancer: association with tumor stage and cyclooxygenase-2.

There is growing body of evidence that post-translational events contribute-in addition to genetic changes-to the progression of malignant tumors. These post-translational alterations may provide targets for new therapeutic approaches. The ELAV-like protein HuR stabilizes a group of cellular mRNAs which contain AU-rich elements in their 3' untranslated region. To investigate a possible contribution of post-translational changes to the progression of colon cancer and to overexpression of COX-2, we studied expression of HuR and COX-2 a cohort of colorectal adenocarcinomas and in colon cancer cell lines. All cell lines showed an expression of HuR mRNA and protein. In tumor tissue of colon carcinomas we observed two different staining patterns of HuR: A nuclear expression in 98% as well as an additional cytoplasmic expression in 53% of cases. COX-2 was expressed in 63% of carcinomas. Cytoplasmic expression of HuR was significantly associated with increased COX-2 expression as well as with high tumor stage. In univariate Kaplan-Meier analysis, grading, tumor stage and nodal status but not HuR or COX-2 expression were prognostic factors for overall survival. Our results suggest that the overexpression of HuR in colon cancer may be part of a regulatory pathway that controls the mRNA stability of cyclooxygenase-2 and provides an interesting example for a contribution of a dysregulation of mRNA stability to the progression of colorectal cancer. Based on our results, further studies are necessary to investigate whether HuR might be a potential target for a molecular tumor therapy.     

Deregulated expression of cell cycle-associated proteins in solid pseudopapillary tumor of the pancreas.

Solid pseudopapillary tumor of the pancreas was studied in a 20-year-old woman and a 54-year-old woman. In the younger patient, the tumor had metastasized to the liver 8 years after distal pancreatectomy. In both neoplasms, the distinct histologic pattern of solid, pseudopapillary, and degenerative cystic areas was present. Analysis by means of immunohistochemistry revealed a diffuse expression for vimentin, neuron-specific enolase, and a focal positivity for al-antitrypsin, whereas epithelial markers were negative in the tumor of the older patient and only focally expressed in the tumor of the younger patient. Immunohistochemical analysis of cell cycle-associated proteins provided an overexpression of cyclin D1 and cyclin D3 in both tumors, although to varying degrees. In addition, the cyclin-dependent kinase inhibitors p21, and to a lesser extent p27, were up-regulated just as mdm2. There was no accumulation of p53 protein, and Ki67-positive cells were extremely scarce. Analysis of the liver metastases showed an immunoreactive profile similar to that of the primary tumor. The results show a deregulation of the cell cycle with overexpression of cell cycle-activating proteins D1 and D3 and a probably counterbalancing upregulation of the cyclin-dependent kinase inhibitors p21 and p27. The findings may explain the low pool of Ki67-reactive tumor cells and the generally good clinical outcome of these tumors. Whether a more profound dysbalance of the cell cycle regulation is responsible for the development of metastatic disease remains to be clarified.     

Expression of alpha-methylacyl-CoA racemase (P504s) in various malignant neoplasms and normal tissues: astudy of 761 cases

Alpha-methylacyl CoA racemase (AMACR), also known as P504S, plays an important role in peroxisomal beta-oxidation of branched-chain fatty acids. It has recently been shown that AMACR is highly expressed in prostate cancer and that it may be an important diagnostic marker for prostate carcinoma. However, little is known about expression of AMACR in normal tissues and other malignant tumors. In this study, we investigated expression of AMACR in 539 malignant tumors and 222 normal human tissues of various types by immunohistochemical analysis. mRNA levels of AMACR in normal organs and in selected tumors were assessed by real time PCR. In normal tissue, high expression of AMACR mRNA was identified in liver, kidney and salivary gland, while AMACR protein was detected in liver (hepatocytes), kidney (tubular epithelial cells), lung (only bronchial epithelial cells), and gallbladder (only mucosal epithelial cells). High expression of AMACR mRNA was found in prostate, liver, and kidney cancers but rarely in stomach and bladder cancers. A high percent of adenocarcinomas arising from these organs express AMACR, including 17 of 21 (81%) of hepatocellular carcinomas and 18 of 24 (75%) of renal cell carcinomas. In addition, carcinomas arising from tissues normally not expressing AMACR were also positive for the antigen, including 17 of 18 (94%) prostate carcinomas, 9 of 29 (31%) of urothelial carcinomas, and 4 of 15 (27%) of gastric adenocarcinomas. Two hundred and fifty cases of adenocarcinomas from lung, breast, pancreas, bile duct, adrenal gland, salivary gland, ovary, thyroid and endometrium were negative or rarely positive for AMACR. Neuroendocrine carcinomas rarely expressed AMACR. Melanomas, squamous cell carcinomas, basal cell carcinomas, soft tissue tumors (including epithelioid sarcomas and synovial sarcoma), thymomas, and germ cell tumors were negative for AMACR. Our data provide important baseline information for using AMACR in clinical practice and also are valuable in furthering understanding of the pathogenic role of AMACR in malignant neoplasms.     

Differential expression of glu-tubulin in relation to mammary gland disease

Tubulin is one of the molecular components that regulate cytosketal structure relating to cell differentiation, invasion, and metastasis in cancer. Recently, glu-tubulin, in which the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidase, overexpression has been reported in malignant tumors of the mammary gland immunohistochemically. We identified 147 cases accessioned in the Department of Pathology, International University of Health and Welfare Hospital from 2003 to 2009. Of the 78 malignant tumor cases, staining for glu-tubulin was observed in 56 (71.8%), and only 22 cases showed no significant staining. However, in benign disease, glu-tubulin staining was detected in the cytoplasm of fibroblasts and endothelial cells, but was completely absent from epithelial cells in 64 of 69 cases. When the expression of glu-tubulin was compared between malignant tumor, benign tumor, and other benign disease, a significant differentiation was found among expressions of this protein. These results indicate that glu-tubulin represents a strong selective expression for cancer cells and may be useful to identify and quantify human breast cancer.     

Response gene to complement 32 (RGC-32) expression on M2-polarized and tumor-associated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4

Response gene to complement 32 (RGC-32) is a cell cycle regulator involved in the proliferation, differentiation and migration of cells and has also been implicated in angiogenesis. Here we show that RGC-32 expression in macrophages is induced by IL-4 and reduced by LPS, indicating a link between RGC-32 expression and M2 polarization. We demonstrated that the increased expression of RGC-32 is characteristic of alternatively activated macrophages, in which this protein suppresses the production of pro-inflammatory cytokine IL-6 and promotes the production of the anti-inflammatory mediator TGF-β. Consistent with in vitro data, tumor-associated macrophages (TAMs) express high levels of RGC-32, and this expression is induced by tumor-derived ascitic fluid in an M-CSF- and/or IL-4-dependent manner. Collectively, these results establish RGC-32 as a marker for M2 macrophage polarization and indicate that this protein is a potential target for cancer immunotherapy, targeting tumor-associated macrophages.     

Role of response gene to complement 32 in diseases

The role of response gene to complement (RGC)-32 as a cell cycle regulator has been attributed to its ability to activate cdc2 kinases and to induce S-phase entry and mitosis. However, recent studies revealed novel functions for RGC-32 in diverse processes such as cellular differentiation, inflammation, and fibrosis. Besides responding to C5b-9 stimulation, RGC-32 expression is also induced by growth factors, hormones, and cytokines. Transforming growth factor β activates RGC-32 through Smad and RhoA signaling, thus initiating smooth muscle cell differentiation. Accumulating evidence has drawn attention to the deregulated expression of RGC-32 in human malignancies, hyper-immunoglobulin E syndrome, and fibrosis. RCG-32 expression is up-regulated in cutaneous T cell lymphoma and colon, ovarian, and breast cancer, but down-regulated in invasive prostate cancer, multiple myeloma, and drug-resistant glioblastoma. A better understanding of the mechanism by which RGC-32 contributes to the pathogenesis of these diseases will provide new insights into its therapeutic potential. In this review we provide an overview of this field and discuss the most recent research on RGC-32.     

GPI-specific phospholipase D mRNA expression in tumor cells of different malignancy

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a highly specific enzyme whose only known substrate is the GPI anchor of cell surface proteins. GPI-PLD measurements, however, are technically difficult since the enzyme is expressed at low levels in cells and tissues, and serum contains large amounts of inactive, latent GPI-PLD interfering with protein-based assays. We have therefore developed a semi-quantitative RT-PCR method to measure mRNA expression of all known GPI-PLD isoforms in cells and tissues. In human ovarian cancer cell lines, GPI-PLD mRNA expression correlated with GPI-PLD enzyme activity and with the shedding of the GPI-anchored tumor and prognostic markers, urokinase receptor and CA125, from the cell surface. This supports a potential role for this enzyme in the generation of circulating prognostic markers in malignant tumors. Similarly, in human epithelial cells of the skin, GPI-PLD mRNA expression increased with tumor progression. Whereas normal keratinocytes did not express significant amounts of GPI-PLD mRNA, expression was dramatically induced by serum in immortalized HaCaT keratinocytes and constitutively high and independent of serum in tumorigenic A431 epidermoid carcinoma cells. In addition, GPI-PLD expression was significantly increased in highly malignant, H-ras-transfected murine bladder carcinoma cells as compared to the low malignant, non-transfected parental cells. The competitive RT-PCR described here represents the first quantitative assay specific for cellular GPI-PLD isoforms, and our in vitro analyses suggest that GPI-PLD expression might be associated with tumor malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Localization of urokinase-type plasminogen activator in stromal cells in adenocarcinomas of the colon in humans.

Human colon adenocarcinomas and adjacent normal colon tissues were stained immunohistochemically with three different monoclonal antibodies and one preparation of polyclonal antibodies against each of the two plasminogen activators, uPA (urokinase type) and tPA (tissue type). The staining patterns seen with the respective sets of antibodies were identical. In all of 10 cases, staining for uPA in the normal colon tissue was confined to scattered fibroblastlike cells in the lamina propria. Other cells, including epithelial and endothelial cells, were uPA negative. All the tumor infiltrates contained many more uPA-positive cells than the normal tissues, but the staining was confined to fibroblastlike cells and endothelial cells in the tumor stroma, while no staining of the malignant epithelial cells was detected. Analysis for uPA by enzyme-linked immunosorbent assay (ELISA) in four cases showed an average uPA content of 0.15 ng uPA/mg protein in the normal colon tissues and 1.6 ng uPA/mg protein in the tumors. Tissue-type plasminogen activator immunoreactivity was confined to endothelial cells in both the normal colon tissue and in the colon carcinomas. These findings may indicate that colon cancer cells recruit stromal cells to produce uPA involved in degradation of the extracellular matrix during invasive growth.     

HIP/PAP, a member of the reg family, is expressed in glucagon-producing enteropancreatic endocrine cells and tumors

Hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) protein, a member of the reg family, is constitutively expressed by some specialized epithelial cell subsets in the digestive tract and the pancreas. We performed a detailed analysis of the expression of HIP/PAP protein in normal digestive endocrine cells according to their localization, lineage, and differentiation stage, and in digestive endocrine tumors according to their site of origin and hormonal profile. In both adult and fetal normal tissues, HIP/PAP expression was detected only in endocrine cells of the small intestine, ascending colon, and pancreas. Two different expression patterns were identified: (a) a strong cytoplasmic labeling observed in the endocrine cells of the digestive mucosa and the outer rim of Langerhans islets specialized in the synthesis of glucagon and glucagon-like peptides; (b) a weak cytoplasmic immunoreactivity observed in the other pancreatic endocrine cell populations. HIP/PAP expression was detected in 36 of the 184 cases of digestive endocrine tumors examined; 32 of these cases (89%) were pancreatic. The 2 patterns observed in the normal state were retained: (a) a strong labeling was observed in 5% to 100% of tumor cells in 26 tumors, all expressing glucagon or glucagon-like peptides; (b) a weak labeling was present in 10 tumors, presenting various hormonal profiles. In conclusion, a strong expression of HIP/PAP is characteristic of glucagon-producing normal and neoplastic enteropancreatic endocrine cells. Our results lend further support to the concept that members of the reg family play regulatory roles in various endocrine cell populations and that their expression in endocrine cells is lineage-specific.