Isolation and characterization of a novel bladder cancer cell line: Inhibition by epidermal growth factor

Summary A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells’ proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.     

Mutant p53R175H upregulates Twist1 expression and promotes epithelial–mesenchymal transition in immortalized prostate cells

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 ‘gain of function'' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53^R175H mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53^R175H, was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial–mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53^R175H mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53^R175H mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.     

Karyotypic changes associated with spontaneous acquisition and loss of tumorigenicity in a human transformed bronchial epithelial cell line: Evidence for In vivo selection of transformed clones

Summary In this study, we describe the karyotypic changes associated with the spontaneous acquisition of tumorigenicity in an immortalized tumor bronchial cell line. Neoplastic transformation of the NL20 human bronchial epithelial cell line occurred after 3 yr in culture, and was associated with loss of chromosome 18 together with acquisition of multiple copies of 9q21.2→34. The nontumorigenic NL20 cell line had been established by transfection of human bronchial epithelial cells with the SV40 T antigen, and had retained a relatively stable karyotype after the first 32 passages in vitro. However, when cells from p184 were inoculated into nude mice, a transplantable tumor was obtained that was derived from a minor clone present in this otherwise stable line. Subsequent passaging of the NL20 cells in vitro did not yield further tumors, and the minor clone from which the tumorigenic NL20T cell line derived was no longer evident in NL20 cells by Passage 205. Furthermore, the original tumorigenic NL20T cells lost the neoplastic phenotype after 25 passages in vitro and reverted to the nontumorigenic karyotype observed at p189. In contrast to the loss of the tumorigenic phenotype and karyotype, which occurred with in vitro passaging of the original tumor, when the NL20T cells were passaged in other nude mice, they continued to give rise to tumors with sevenfold amplifications of 9q sequences and loss of chromosome 18, and cells from the secondary tumors (NL20T-A cells) have maintained a stable karyotype and remain tumorigenic even after 64 passages in vitro. A mixture of 10% tumorigenic NL20T-A and 90% nontumorigenic NL20 cells formed tumors in athymic nude mice when cultured in vitro on fibronectin, but not on plastic; cytogenetic analysis demonstrated that the tumors and cell cultures were composed of tumorigenic NL20T-A cells, whereas cytogenetic analysis of cells cultured on plastic were identical to the nontumorigenic NL20 cells. These data support the hypothesis that neoplastic transformation in our original cell line arose from in vivo selection of a small mutant clone, which had arisen in culture and was subsequently selected in vivo but was lost with in vitro culture.     

Establishment and characterization of an osteopontin-null cutaneous squamous cell carcinoma cell line

Osteopontin (OPN) is a secreted glycoprotein implicated to function in cancer development and metastasis. Although elevated expression of OPN are observed in cancer cells of various types, in some cases, only the cells in the stromal region surrounding the tumor express OPN, suggesting distinct functional roles for this protein derived from host cells and from cancer cells. To provide a model for addressing the functions and mechanisms of host-derived OPN in cancer progression and metastasis, a cutaneous squamous cell carcinoma cell line (ONSC) that lacks the OPN gene, Spp1, was established. This line of cells was derived from a squamous cell carcinoma that developed in a female, OPN-null mouse subjected to two-stage skin carcinogenesis. Morphologically, ONSC cells resemble epithelial cells, and they express the epithelial markers, K1, K14, and p63, as confirmed by immunohistochemical analyses. Genomic analyses indicate the presence of mutated H-Ras and p53 genes. ONSC cells form colonies in soft agar and, subcutaneously injected into athymic nude mice, develop into squamous cell carcinomas that metastasize to the lungs. Lacking OPN expression, these squamous cell carcinoma cells provide a model to address the function of host OPN in the context of cancer progression and metastasis.     

Epithelial-mesenchymal transition of ovarian tumor cells induces an angiogenic monocyte cell population

Vasculogenesis, or recruitment of progenitors able to differentiate into endothelial-like cells, may provide an important contribution to neovessel formation in tumors. However, the factors involved in the vasculogenic process and in particular the role of the epithelial-mesenchymal transition of tumor cells have not yet been investigated. We found a CD14⁺/KDR⁺ angiogenic monocyte population in undifferentiated ovarian tumors, significantly increased in the corresponding tumor metastasis. In vitro, monocyte differentiation into CD14⁺/KDR⁺ cells was induced by conditioned media from the primary ovarian tumor cells expressing a mesenchymal phenotype. In contrast, the ovarian tumor cell line SKOV3 expressing an epithelial phenotype was unable to stimulate the differentiation of monocytes into CD14⁺/KDR⁺ cells. When an epithelial-mesenchymal transition was induced in SKOV3, they acquired this differentiative ability. Moreover, after mesenchymal transition pleiotrophin expression by SKOV3 was increased and conversely its blockade significantly reduced monocyte differentiation. The obtained CD14⁺/KDR⁺ cell population showed the expression of endothelial markers, increased the formation of capillary-like structures by endothelial cells and promoted the migration of ovarian tumor cells in vitro. In conclusion, we showed that the epithelial-mesenchymal transition of ovarian tumor cells induced differentiation of monocytes into the pro-angiogenic CD14⁺/KDR⁺ population and thus it may provide a tumor microenvironment that favours vasculogenesis and metastatization of the ovarian cancer.     

Adenovirus Serotype 5 Variants Defective in Early Genes: Selective Replication in p53-Deficient Human Tumor Cells

Site-directed mutagenesis was used to construct human adenovirus serotype 5 (Ad5) variants defective in E1A or E1B. Mutant Adel3 with deletion from E1A was markedly attenuated in permissive cell cultures regardless of the p53 status, and replicated efficiently only in cells of the complementing 293 line. Mutant Adel2 with deletion from E1B55K infected the 293 line cells and p53-deficient human tumor cells (A431, SW480, HEp2) with efficiencies similar to those of Ad5, whereas its replication in normal p53-positive cells was substantially limited. Thus, Adel2 proved to be capable of selective infection and lysis of p53-deficient human tumor cells in vitro. On intratumor injection, Adel2 dramatically suppressed the growth of human epidermoid carcinoma (A431) in nude mice. Adel2 is thus a promising model for designing therapeutic agents against p53-deficient human tumors.     

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.     

Inhibition of Breast Cancer Metastasis Suppressor 1 Promotes a Mesenchymal Phenotype in Lung Epithelial Cells That Express Oncogenic K-RasV12 and Loss of p53

Expression of the breast cancer metastasis suppressor 1 (BRMS1) protein is dramatically reduced in non-small cell lung cancer (NSCLC) cells and in primary human tumors. Although BRMS1 is a known suppressor of metastasis, the mechanisms through which BRMS1 functions to regulate cell migration and invasion in response to specific NSCLC driver mutations are poorly understood. To experimentally address this, we utilized immortalized human bronchial epithelial cells in which p53 was knocked down in the presence of oncogenic K-Ras^V12 (HBEC3-p53KD-K-Ras^V12). These genetic alterations are commonly found in NSCLC and are associated with a poor prognosis. To determine the importance of BRMS1 for cytoskeletal function, cell migration and invasion in our model system we stably knocked down BRMS1. Here, we report that loss of BRMS1 in HBEC3-p53KD-K-Ras^V12 cells results in a dramatic increase in cell migration and invasion compared to controls that expressed BRMS1. Moreover, the loss of BRMS1 resulted in additional morphological changes including F-actin re-distribution, paxillin accumulation at the leading edge of the lamellapodium, and cellular shape changes resembling mesenchymal phenotypes. Importantly, re-expression of BRMS1 restores, in part, cell migration and invasion; however it does not fully reestablish the epithelial phenotype. These finding suggests that loss of BRMS1 results in a permanent, largely irreversible, mesenchymal phenotype associated with increased cell migration and invasion. Collectively, in NSCLC cells without p53 and expression of oncogenic K-Ras our study identifies BRMS1 as a key regulator required to maintain a cellular morphology and cytoskeletal architecture consistent with an epithelial phenotype.     

Demonstration of Ia antigens on mouse intestinal epithelial cells by immunoferritin labeling

Several kinds of epithelial cells that express H-2 antigens were studied by immunoferritin labeling with an antiserum reacting only with antigens of theI region of theH-2 complex. Spleen lymphocytes were used to test the labeling system and the effect of the epithelial cell dissociation procedure on Ia antigens. Immunoglobulin-positive B10.BR lymphocytes were labeled with an anti-la^k serum (A.TH anti-A.TL serum absorbed with BALB/c and B10.D2 cells), while congenic B10.D2 lymphocytes were unlabeled. The distribution of labeled Ia antigens on living B10.BR lymphocytes was patchy, while on cells fixed in periodate-lysine-paraformaldehyde before labeling, the distribution of label was continuous. Fixation evidently immobilized Ia antigens in the lymphocyte membrane. Trypsin and collagenase, as used in the epithelial cell dissociation procedure, had no discernible effect on the Ia antigens of lymphocytes. The epithelial cells studied included the columnar absorptive cells of the small intestine, uterine lining epithelium, tracheal brush cells, and pancreatic exocrine and duct cells. These cells were fixed before dissociation from their respective tissues. Ia antigens were detected only on the columnar absorptive cells of the small intestine. These cells labeled equally well with an antiserum reacting only with theK -end of theH-2 complex. In both cases, congenic control intestinal cells were unlabeled. Thus, intestinal epithelial cells appear to express theIa, K, and presumablyD regions of theH-2 complex, while the other epithelial cell types express only the K and D antigens. On fixed intestinal epithelial cells, Ia and H-2K antigens were continuously distributed on the lateral and basal cell membranes including the zonula adherens, but the antigens were absent from the apical microvillous membrane and the zonula occludens.     

Mouse thymic epithelial cell lines expressing "Aire" and peripheral tissue-specific antigens reproduce in vitro negative selection of T cells

In the human thymus, AIRE (autoimmune regulator) gene is expressed in a very limited type of medullary thymic epithelial cells (mTECs) and no cognate cell lines are available, hence the molecular analysis of AIRE gene function has been difficult. To improve this situation, we attempted to isolate Aire-expressing cells and established three cell lines (Aire⁺TEC1, Aire⁺TEC2, Aire⁺DC) from the abnormally enlarged thymus, which was developed in the transgenic mice expressing SV40 T-antigen driven by the mouse Aire gene promoter. When these Aire⁺ cell lines were co-cultured with fresh thymocytes, they adhered to the majority of thymocytes and induced apoptosis as if negative selection of T-cells in the thymus is occurring in vitro. Further analysis revealed that these Aire⁺ cell lines are derived from mTECs and exhibit characteristic natures of “antigen presenting cells” including several distinct abilities: to express a variety of peripheral tissue-specific antigens, to produce immunoproteasome and immunological synapse, and to express some of TNFSFs (tumor necrosis factor super families). Thus, the newly established Aire⁺ cell lines will be invaluable for the further detailed analysis of AIRE gene function in the central tolerance of immunity and autoimmune disease.     

Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules

 The adoptive transfer of tumor-draining lymph node (LN) T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56 ^lck and p59 ^fyn and proteins of the NF-κB family were analyzed in tumor-draining LN T cells. The levels of p56 ^lck and p59 ^fyn were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56 ^lck and p59 ^fyn and protein tyrosine kinase activity increased. Interestingly, the levels of p56 ^lck , p59 ^fyn , and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear κB DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-κB. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function. Received: 28 August 1997 / Accepted: 23 February 1998     

Isolation of a stromal cell line from an early passage of a mouse mammary tumor line: a model for stromal parenchymal interactions

We have developed a murine mammary tumor cell line, MC4-L4, and after 15 passages, a spindle-shaped population became evident. The cuboidal cells, MC4-L4E, cloned by limit dilution, proved to be epithelial tumor cells. When inoculated in syngeneic mice, they gave rise to invasive metastatic carcinomas expressing estrogen and progesterone receptors. These tumors regressed after anti-progestin treatment and stopped growing after 17-beta-estradiol administration. In vitro, they were insensitive to medroxyprogesterone acetate (MPA), 17-beta-estradiol, and EGF and were inhibited by TGFbeta1. They expressed mutated p53 and estrogen receptors alpha; progesterone receptors were undetectable. Cells were polyploid and shared the same four common marker chromosomes present in the parental tumor in addition to an exclusive marker. Spindle-shaped cells, MC4-L4F, were selected by differential attachment and detachment and proved to be non-epithelial non-tumorigenic cells. They were cytokeratin negative, showed mesenchymal features by electron microscopy, differentiated to adipocytes when treated with an adipogenic cocktail, were stimulated by TGFbeta1 and EGF, showed a wild-type p53, and did not exhibit the marker chromosomes of the parental tumor. Although they expressed estrogen receptors alpha, they were insensitive to 17-beta-estradiol in proliferation assays. Co-cultures of both cell types had a synergic effect on progesterone receptors expression and on cell proliferation, being the epithelial cells, the most responsive ones, and 17-beta-estradiol increased cell proliferation only in co-cultures. Cytogenetic studies and data on p53 mutations rule out the possibility of an epithelial mesenchymal transition. Their unique characteristics make them an excellent model to be used in studies of epithelial-stromal interactions in the context of hormone responsiveness in hormone related tumors.     

TNFalpha up‐regulates SLUG via the NF‐kappaB/HIF1alpha axis,which imparts breast cancer cells with a stem cell‐like phenotype

Extracellular and intracellular mediators of inflammation, such as tumor necrosis factor alpha (TNFα) and NF‐kappaB (NF‐κB), play major roles in breast cancer pathogenesis, progression and relapse. SLUG, a mediator of the epithelial–mesenchymal transition process, is over‐expressed in CD44⁺/CD24^? tumor initiating breast cancer cells and in basal‐like carcinoma, a subtype of aggressive breast cancer endowed with a stem cell‐like gene expression profile. Cancer stem cells also over‐express members of the pro‐inflammatory NF‐κB network, but their functional relationship with SLUG expression in breast cancer cells remains unclear. Here, we show that TNFα treatment of human breast cancer cells up‐regulates SLUG with a dependency on canonical NF‐κB/HIF1α signaling, which is strongly enhanced by p53 inactivation. Moreover, SLUG up‐regulation engenders breast cancer cells with stem cell‐like properties including enhanced expression of CD44 and Jagged‐1 in conjunction with estrogen receptor alpha down‐regulation, growth as mammospheres, and extracellular matrix invasiveness. Our results reveal a molecular mechanism whereby TNFα, a major pro‐inflammatory cytokine, imparts breast cancer cells with stem cell‐like features, which are connected to increased tumor aggressiveness. J. Cell. Physiol. 225: 682–691, 2010. © 2010 Wiley‐Liss, Inc.     

Hypoxia Induces EMT in Low and Highly Aggressive Pancreatic Tumor Cells but Only Cells with Cancer Stem Cell Characteristics Acquire Pronounced Migratory Potential

Tumor hypoxia induces epithelial-mesenchymal transition (EMT), which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Whether EMT generates CSCs de novo, enhances migration of existing CSCs or both is unclear. We examined patient tissue of pancreatic ductal adenocarcinoma (PDA) along with carcinomas of breast, lung, kidney, prostate and ovary. For in vitro studies, five established PDA cell lines classified as less (CSC^low) and highly aggressive CSC-like cells (CSC^high) were examined by single and double immunofluorescence microscopy, wound-, transwell-, and time-lapse microscopy. HIF-1α and Slug, as well as HIF-2α and CD133 were co-expressed pointing to a putative co-existence of hypoxia, EMT and CSCs in vivo. CSC^high cells exhibited high basal expression of the mesenchymal Vimentin protein but low or absent expression of the epithelial marker E-cadherin, with the opposite result in CSC^low cells. Hypoxia triggered altering of cell morphology from an epithelial to a mesenchymal phenotype, which was more pronounced in CSC^high cells. Concomitantly, E-cadherin expression was reduced and expression of Vimentin, Slug, Twist2 and Zeb1 enhanced. While hypoxia caused migration in all cell lines, velocity along with the percentage of migrating, polarized and pseudopodia-forming cells was significantly higher in CSC^high cells. These data indicate that hypoxia-induced EMT occurs in PDA and several other tumor entities. However although hypoxia-induced EMT signaling occurs in all tumor cell populations, only the stem-like cells acquire high migratory potential and thus may be responsible for invasion and metastasis.