Immortalized ovarian surface epithelial cells acquire tumorigenicity by Acrogranin gene overexpression

Malignant transformation is caused by multi-step genetic mutations, and growth factors are believed to play important roles in developing and maintaining malignant phenotype. However, there is no direct evidence that a specific growth factor contributes to malignant transformation of phenotypically normal cells. In order to assess the function of Acrogranin (also known as granulin epithelial precursor; GEP) in ovarian carcinogenesis, ovarian surface epithelial (OSE) cells, which are supposed to be the origin of primary ovarian epithelial cancer, were transfected with combined genes of hTERT, SV40 LT, and Acrogranin. Introduction of hTERT and SV40 LT was sufficient for immortalizing OSE cells but not enough for tumor formation in nude mice. In contrast, transfection and overexpression of Acrogranin in immortalized OSE cells showed augmented clonogenicity in soft agar and obvious tumorigenicity in nude mice. This is the first study showing evidence that a specific growth factor plays a direct role in malignant transformation in ovarian cancer development.     

Multipotential behaviour of cloned rat mesothelioma cells with epithelial phenotype

Reference cultures derived from a transplantable rat mesothelioma were obtained by cloning cells three times in soft agar. Each line, designated "CARM-Lines", was selected on the basis of their epithelial or fibroblastic phenotype, and their uniform morphology. Three epithelial lines were used for more detailed in vitro studies comparing morphological and biological criteria at early and late passages. All three lines exhibited both epithelial and fibroblastic elements after 10-14 passages in vitro, demonstrating that the dimorphic histology of these tumours could be derived from a single aberrant cell. Morphology and growth characteristics of these cells were density-dependent. Anchorage dependent and independent clonogenic assays did not correlate. Anchorage dependent colony formation was the only parameter which differed markedly from the original parent line in the assays described. In vivo evidence of chondrogenesis and attempted ossification support the concept of a multipotential cell contributing to the diverse primary tumour morphology by cellular modulation or differentiation.     

Hypodontia phenotype in patients with epithelial ovarian cancer

Background

Ovarian cancer is usually diagnosed in an advanced stage and the present clinical and diagnostic molecular markers for early OC screening are insufficient. The aim of this study was to identify potential relationship between the hypodontia and epithelial ovarian cancer (EOC).

Patients and methods

A retrospective study was conducted on 120 patients with EOC treated at the Department of Gynaecologic and Breast Oncology at the University Clinical Centre and 120 gynaecological healthy women (control group) of the same mean age. Women in both groups were reviewed for the presence of hypodontia and the patients with EOC also for clinicopathological characteristics of EOC according to hypodontia phenotype.

Results

Hypodontia was diagnosed in 23 (19.2%) of patients with EOC and 8 (6.7%) controls (p = 0.004; odds ratio [OR] = 3.32; confidence interval [CI], 1.42–7.76). There was no statistically significant difference in patients with EOC with or without hypodontia regarding histological subtype (p = 0.220); they differed in regard to FIGO stage (p = 0.014; OR =3.26; CI, 1.23–8.64) and tumour differentiation grade (p = 0.042; OR = 3.1; CI, 1.01–9.53). Also, bilateral occurrence of EOC was more common than unilateral occurrence in women with hypodontia (p = 0.021; OR = 2.9; CI, 1.15–7.36). We also found statistically significant difference between the ovarian cancer group and control group in presence of other malignant tumours in subjects (p < 0.001).

Conclusions

The results of the study suggest a statistical association between EOC and hypodontia phenotype. Hypodontia might serve as a risk factor for EOC detection.     

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness.     

Clonal analysis of tumorigenicity and paratumorigenic phenotypes in rat liver epithelial cells chemically transformed in vitro

Clonal subpopulations of a chemically induced tumorigenic rat liver epithelial cell line were analyzed for their cellular, biochemical, and in vitro growth properties and their tumorigenicity after injection into day-old newborn isogeneic rats. The phenotypic properties studied included DNA content; growth rate in culture; activities of gamma-glutamyl transpeptidase, NADH diaphorase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase; ability to grow in calcium-poor medium; and ability to form colonies in soft agar. The results show that none of these phenotypes cosegregates with tumorigenicity and therefore is not reliable as a "marker" phenotype for neoplastic transformation in cultured rat liver epithelial cells. The poor correlations, either qualitatively or quantitatively, between paratumorigenic phenotypes and tumorigenicity suggest that neoplastic transformation in these cells involves a specific transforming gene locus or loci and that in vitro paratumorigenic phenotypes are merely epiphenomena of neoplastic transformation and progression. This study further reveals that the efficiency of the tumorigenicity assay of cultured rat liver epithelial cells in isogeneic newborn rats can be considerably improved by incubating the cells in medium containing only trace amounts of serum prior to transplantation into the host animals.     

Dynamic alterations of the extracellular environment of ovarian surface epithelial cells in premalignant transformation,tumorigenicity, and metastasis

BACKGROUND: Ovarian surface epithelial cells are positionally organized as a single cell layer by a sheet of basement membrane. It is believed that the contact of the ovarian surface epithelial cells with the basement membrane regulates cell growth and ensures the organization of the epithelium. Disabled-2 (Dab2), a signal transduction protein and a candidate tumor suppressor of ovarian carcinoma, functions in positional organization of ovarian surface epithelial cells. In ovarian carcinomas, genetic and epigenetic changes enable the tumor cells to escape positional control and proliferate in a disorganized fashion. Alterations in the extracellular environment may also be critical for tumor initiation and progression. METHODS: We analyzed and compared the presence of collagen IV and laminin, the scaffold proteins of the basement membrane, and Dab2 in 50 ovarian tumors that are restricted to the ovaries and in 50 metastases of ovarian tumors by immunohistochemistry. Expression of collagen IV, laminin, and Dab2 was also analyzed by Northern blotting in a panel of human ovarian surface epithelial and cancer cell lines. RESULTS: The basement membrane is often absent in morphologically benign ovarian surface and cyst epithelium and low-grade tumors and collagen IV and laminin are absent in the extracellular matrix of most of the primary tumors tested. Of the 50 ovarian tumors confined to the ovaries, 6% (3 of 50) were collagen IV positive and 24% (12 of 50) were laminin positive tumors. Of the 50 metastatic tumors, 16% (8 of 50) are collagen IV positive and 86% (43 of 50) are laminin positive. In addition, even in the metastatic ovarian tumors that are largely collagen IV negative, there are pockets of local areas in which the tumor cells are surrounded by collagen IV-positive staining. Dab2 is absent in the majority of ovarian tumors found in both ovaries and metastatic sites. In both nontumorigenic human ovarian surface epithelial and cancer cell lines, collagen IV, laminin, and Dab2 are expressed aberrantly. CONCLUSIONS: Loss of the basement membrane may be an early event in the preneoplastic transformation of ovarian surface epithelium and in the early stages of tumorigenesis before tumor invasion and metastasis. The majority of primary ovarian tumors examined lack collagen IV and laminin in their extracellular matrix. However, expression of laminin is restored in the majority of metastatic tumors. Reexpression of collagen IV may also contribute to tumor metastasis. The ability of tumor cells to dynamically alter the expression of collagen IV and laminin may facilitate the shedding of cancer cells into the peritoneal spaces and subsequent attachment to the metastatic sites. We propose that loss of collagen IV and laminin may be an initial event in ovarian tumorigenicity and that restoration of collagen IV and laminin expression in the later stages of tumor development may promote metastasis of ovarian tumors.     

Androgen-induced differentiation and tumorigenicity of human prostate epithelial cells

Androgen ablation is the primary treatment modality for patients with metastatic prostate cancer; however, the role of androgen receptor signaling in prostate cancer development remains enigmatic. Using a series of genetically defined immortalized and tumorigenic human prostate epithelial cells, we found that introduction of the androgen receptor induced differentiation of transformed prostate epithelial cells to a luminal phenotype reminiscent of organ-confined prostate cancer when placed in the prostate microenvironment. Moreover, androgen receptor expression converted previously androgen-independent, tumorigenic prostate epithelial cells into cells dependent on testosterone for tumor formation. These observations indicate that androgen receptor expression is oncogenic and addictive for the human prostate epithelium.     

正常卵巢上皮细胞和卵巢上皮癌细胞基因表达差异的研究

目的：探讨卵巢上皮癌细胞相关基因的差异表达。方法：采用舍384条肿瘤相关基因的cDNA阵谱检测了卵巢上皮癌细胞株SKOV-3及正常卵巢上皮细胞的基因谱，分析卵巢上皮癌细胞相关基因的差异表达。结果：在384条候选基因中，与卵巢癌相关的差异表达基因33条，其中22条表达上调，11条表达下调。结论：cDNA阵谱技术是筛查卵巢癌相关基因的有效方法。     

Membrane-type-1 matrix metalloproteinase confers tumorigenicity on nonmalignant epithelial cells

Overexpression of membrane-type-1 matrix metalloproteinase (MT1-MMP) in tumor cells has previously been shown to enhance tumor growth and metastasis. To establish if MT1-MMP is also able to confer tumorigenicity on nonmalignant epithelial cells, we transfected human MT1-MMP cDNA into Madin-Darby canine kidney (MDCK) cells expressing a tetracycline-repressible transactivator. Induction of MT1-MMP in the absence of doxycycline (Dox) was associated with activation of exogenous MMP-2 as well as with formation of large cysts and increased invasiveness in collagen matrices. Transfected cells were inoculated subcutaneously into two groups of nude mice, one of which received Dox to inhibit expression of MT1-MMP. Formation of tumor xenografts was observed in 11 of 17 mice maintained without Dox, but only in two of nine mice that received Dox (P<0.05). The xenografts were composed of tubular structures interspersed within a highly cellular stroma. The epithelial cells delimiting the lumen were polarized, as indicated by the basolateral distribution of Na,K-ATPase. Despite their differentiated appearance, the tumors lacked a well-defined boundary, and epithelial tubules invaded adjacent muscular layers. These results demonstrate that conditional expression of MT1-MMP in nonmalignant MDCK epithelial cells is by itself sufficient to drive formation of invasive tumors.     

Syndecan-1 promotes the angiogenic phenotype of multiple myeloma endothelial cells

Angiogenesis is considered a hallmark of multiple myeloma (MM) progression. In the present study, we evaluated the morphological and functional features of endothelial cells (ECs) derived from bone marrow (BM) of patients affected by MM (MMECs). We found that MMECs compared with normal BM ECs (BMECs) showed increased expression of syndecan-1. Silencing of syndecan-1 expression by RNA interference technique decreased in vitro EC survival, proliferation and organization in capillary-like structures. In vivo, in severe combined immunodeficient mice, syndecan-1 silencing inhibited MMEC organization into patent vessels. When overexpressed in human umbilical vein ECs and BMECs, syndecan-1 induced in vitro and in vivo angiogenic effects. Flow-cytometric analysis of MMECs silenced for syndecan-1 expression indicated a decreased membrane expression of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2). Immunoprecipitation and confocal analysis showed colocalization of VEGFR-2 with syndecan-1. Absence of nuclear translocation of VEGFR-2 in syndecan-1-knockdown cells together with the shift from perinuclear localization to recycling compartments suggest a role of syndecan-1 in modulation of VEGFR-2 localization. This correlated with an in vitro decreased VEGF-induced invasion and motility. These results suggest that syndecan-1 may contribute to the highly angiogenic phenotype of MMECs by promoting EC proliferation, survival and modulating VEGF-VEGFR-2 signalling.     

Transformation of cultured rat ovarian surface epithelial cells by Kirsten murine sarcoma virus

A method is described for the culture of rat ovarian surface epithelial cells, i.e., the cellular component thought to be the source of most ovarian cancers. These cells in culture have a characteristic epithelial morphology which distinguishes them from other ovarian cell types. Cultured surface epithelial cells are histochemically positive for 17 beta-hydroxysteroid dehydrogenase and negative for delta 5-3 beta-hydroxysteroid dehydrogenase, the same as in cryostat sections of whole rat ovary. Ultrastructurally, cultured surface epithelial cells have basement membranes, microvilli, and apical intercellular junctions. Kirsten murine sarcoma virus was used to produce three transformed cell lines from pure first-passage cultures of these cells. These three lines retained 17 beta-hydroxysteroid dehydrogenase activity and showed slight delta-3 beta-hydroxysteroid dehydrogenase activity. Tumors resulting when these cells were injected s.c. or i.p. into immunosuppressed female rats were highly malignant, resembling histologically human ovarian endometrioid stromal sarcoma. This is the first demonstration of the susceptibility of ovarian surface epithelium to an oncogenic virus.     

Modulation of differentiation in PA-1 human teratocarcinoma cells after N-ras oncogene-induced tumorigenicity.

We have been studying the effect of oncogenes on differentiation using the human ovarian teratoma-derived cell line PA-1. From this study we have characterized variants representing four stages relevant to multistage carcinogenesis, two non-tumorigenic and two tumorigenic. The two non-tumorigenic cell variants differ in that one is resistant to transformation by ras oncogenes whereas the other can be transformed to tumorigenicity. When these non-tumorigenic PA-1 variants are treated with retinoic acid (RA), a morphogen, they stop dividing, begin to express homeobox genes, and change in morphology. Transfection of an activated N-ras oncogene into ras-resistant non-tumorigenic PA-1 cells does not alter the RA responsiveness of the cells, indicating that expression of the activated oncogene is not sufficient for blocking RA-induced differentiation. Spontaneous activation of an N-ras oncogene leading to tumorigenic transformants and gene transfer-induced N-ras transformants are resistant to these effects of RA. However, another spontaneous transformant of PA-1 cells that does not contain an activated N-ras is responsive to RA. We prepared somatic cell hybrids of the RA-non-responsive, N-ras-transformed and tumorigenic PA-1 cell and the RA-responsive, ras-resistant non-tumorigenic PA-1 cell; the hybrid cell lines continue to express the oncogene but are non-tumorigenic. These non-tumorigenic hybrids are responsive to RA with regard to morphological changes, growth arrest and induction of homeobox gene expression. Tumorigenic revertants of these hybrids arise as a result of the loss of some chromosomes; these hybrid cells express the oncogene but have lost RA responsiveness. These results indicate that tumorigenic transformation in general is not sufficient to induce RA resistance, and resistance to differentiation may be oncogene-specific. In addition, the expression of an activated N-ras oncogene alone is insufficient to induce resistance to RA and ras-induced tumorigenicity is necessary. Therefore, some feature of cellular metabolism that is altered by and discordantly segregates with tumorigenic transformation controls responsiveness to RA. This controlling element is presumably a tumor suppressor.     

上皮源性卵巢癌干细胞的研究进展

肿瘤干细胞(cancer stem cells,CSCs)是肿瘤组织内具有自我更新能力以及无限增殖和多向分化潜能的一群细胞,对化疗药物耐受.卵巢癌干细胞(epithelial ovarian cancer stem cells,EOCSCs)在卵巢癌的发生发展、侵袭转移和耐药复发过程中都起到了重要作用.传统的肿瘤细胞减灭术联合顺铂、紫杉醇全身化疗对减小肿瘤体积,缓解临床症状具有一定的作用,但治疗后残留的EOCSCs能够短时间内重建肿瘤组织,是卵巢癌复发和难治的根本原因.深入了解EOCSCs的生物学特性,探索EOCSCs的发生发展机制,研究针对EOCSCs的靶向治疗药物,是抗肿瘤治疗的关键.     

Myofibroblast-induced tumorigenicity of pancreatic ductal epithelial cells is L1CAM dependent

Pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis, representing one risk factor for PDAC, are characterized by a marked desmoplasia enriched of pancreatic myofibroblasts (PMFs). Thus, PMFs are thought to essentially promote pancreatic tumorigenesis. We recently demonstrated that the adhesion molecule L1CAM is involved in epithelial-mesenchymal transition of PMF-cocultured H6c7 human ductal epithelial cells and that L1CAM is expressed already in ductal structures of chronic pancreatitis with even higher elevation in primary tumors and metastases of PDAC patients. This study aimed at investigating whether PMFs and L1CAM drive malignant transformation of pancreatic ductal epithelial cells by enhancing their tumorigenic potential. Cell culture experiments demonstrated that in the presence of PMFs, H6c7 cells exhibit a profound resistance against death ligand-induced apoptosis. This apoptosis protection was similarly observed in H6c7 cells stably overexpressing L1CAM. Intrapancreatic inoculation of H6c7 cells together with PMFs (H6c7co) resulted in tumor formation in 7/8 and liver metastases in 6/8 severe combined immunodeficiency (SCID) mice, whereas no tumors and metastases were detectable after inoculation of H6c7 cells alone. Likewise, tumor outgrowth and metastases resulted from inoculation of L1CAM-overexpressing H6c7 cells in 5/7 and 3/7 SCID mice, respectively, but not from inoculation of mock-transfected H6c7 cells. Treatment of H6c7co tumor-bearing mice with the L1CAM antibody L1-9.3/2a inhibited tumor formation and liver metastasis in 100 and 50%, respectively, of the treated animals. Overall, these data provide new insights into the mechanisms of how PMFs and L1CAM contribute to malignant transformation of pancreatic ductal epithelial cells in early stages of pancreatic tumorigenesis.     

Regulation of exosomes released from normal ovarian epithelial cells and ovarian cancer cells

Tumor Biology - As important cell to cell communicator, exosomes carry a range of bioactive molecules which can significantly influence phenotype of recipient cells. Inhibiting or removing cancer...     

Restoration of an impaired TGF-beta1 autocrine growth-inhibitory circuit results in growth inhibition of ovarian epithelial cancer cells and complete inhibition of their tumorigenicity

The disruption of the transforming growth factor beta1 (TGF-beta1) autocrine growth-suppressive circuit is a major and possibly early event mediating the malignant transformation of normal epithelia. TGF-beta1 is secreted as a latent homodimer-peptide that, upon activation, binds a receptor complex. This in turn activates a signal transduction cascade that results in proliferation inhibition of epithelial cells. The growth-inhibitory pathway can be interrupted at several levels: insufficient secretion and activation of TGF-beta1 ligand, mutational inactivation of the receptors or signal transduction intermediates or at the level of the nuclear effector molecules. We have investigated the effect of restoring the growth-inhibitory autocrine circuit in epithelial cancer cells that have retained sensitivity to growth inhibition by TGF-beta1 but which produce and secrete insufficient amounts of endogenous peptide. These cancer cells were transduced with a recombinant adenovirus containing a TGF-beta1 cDNA driven by a CMV promoter and coding for a constitutively bioactive TGF-beta1 peptide. Restituting the TGF-beta1 autocrine growth-suppressive circuit in these cancer cells had a potent growth-inhibitory effect in vitro. Moreover, in vitro transduced cells lost their tumorigenicity in nude mice. As disruption of TGF-beta's autocrine growth circuit is thought to be an early event in the malignant transformation of several epithelial cancers, early correction of this defect might in the future lead to cancer preventive strategies.     

Effect of reproductive hormones on ovarian epithelial tumors: II. Effect on angiogenic activity

Menstrual cycle activity predisposes to ovarian epithelial tumors based on numerous epidemiological studies. We showed that the hormones involved in menstrual cycle regulation modulate cell cycle activity in these tumors in an accompanying paper. We investigated whether such hormones could also influence angiogenesis, an important determinant of tumor progression, in the same tumors. Treatment with progesterone (P4) stimulated VEGF protein secretion in 4 of 5 ovarian carcinoma cell lines examined. Northern blot analyses performed in MCV50 cells showed that this effect was accompanied by increased VEGF mRNA levels. P4 also stimulated VEGF promoter activity in these cells. Estradiol (E2) showed a similar, but substantially smaller effect on VEGF secretion which was additive to that of P4. Conditioned medium from P4-treated cells strongly stimulated angiogenesis on chicken chorio-allantoic membranes. Incubating the conditioned medium with a neutralizing anti-VEGF antibody, but not with non-specific immunoglobulins abolished this effect. Angiogenic activity was not altered by treatment of the membranes with P4 directly. We conclude that P4 can stimulate angiogenic activity via induction of VEGF secretion in some ovarian epithelial tumors. Therapeutic use of progestins may be most effective when administered in combination with an anti-angiogenic agent, at least against a subset of ovarian carcinomas.     

Surface membrane nucleoside triphosphatase activity and tumorigenicity of cultured liver epithelial cells.

A cell surface-located nucleoside triphosphatase activity can be assayed in liver epithelial cultures in situ with the incubation of intact cells in a medium containing [gamma-32P]adenosine triphosphate and correlated with the tumorigenicity of these cells in neonatal Wistar rats. The ectoenzyme activity of normal diploid cell lines is minimal, whereas a considerably high activity has been found in all tumorigenic cell lines tested. The optimum condition for the adenosinetriphosphatase activity is physiological with regard to osmolarity, ionic composition, pH, and substrate concentration in the medium. The enzyme is significantly stimulated by Ca2+, and its activation is controlled by Mg2+. Histochemical examinations indicate that glutaraldehyde-fixed cells of tumorigenic lines have Ca2+-stimulated adenosinetriphosphatase activity on the external surface. The isotopic assay of adenosine triphosphate hydrolysis by intact cells may provide a rapid method for screening oncogenesis in vitro of liver epithelial cells.     

Cytogenetic instability in ovarian epithelial cells from women at risk of ovarian cancer

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.