Characterization of a cell line (SW756) derived from a human squamous carcinoma of the uterine cervix

Summary An established cell line, SW756, derived from a primary squamous carcinoma of the uterine cervix is described by its morphology, ultrastructure, karyotype, genetic signature analysis, HLA typing, and tumorigenesis in the nude mouse. Cultured cells obtained from the SW756 derived nude mouse tumor also were studied for chromosome and isozyme markers. The original tumor was poorly differentiated carcinoma with minimal keratinization and is compared with that occurring in the nude mouse after the cultured cells were inoculated. The nude mouse tumor showed similar histological features, but better differentiation than the original tumor. Karyotype analysis of SW756 demonstrated a hyperdiploid stem line number and several marker chromosomes (MI-M6). No HeLa marker chromosomes were identified. The isozyme pattern for SW756 reported by others has been confirmed. The unique chromosome and isozyme features have been identified repeatedly in the cultured cells and, most importantly, in the post nude mouse culture. We recommend SW756 as a defined human tumorigenic cell line derived from a primary squamous carcinoma of the uterine cervix. This investigation was supported in part by Public Health Research Grant CA-06294 from the National Cancer Institute, Department of Health and Human Services.     

Establishment and characterization of a new SCC antigen producing cell line (HCS-2) from a carcinoma of the uterine cervix

The cell line designated HCS-2 established from a squamous cell carcinoma of the uterine cervix has been subcultivated 77 times since Nov. 8, 1983. The cultured cells appear epithelial in shape, with a pavement-like arrangement and grow without contact inhibition. In electron microscopy, the cells are characterized by desmosomal cell contacts and a few tonofilaments. The cells are transplanted subcutaneously to nude mice and produce tumor which resembles the original tumor of large cell non-keratinizing squamous cell carcinoma. The growth rate of subculture has increased gradually, and population doubling time of cells at 17th passage was about 65 hours. The chromosome studies show aneuploidy and chromosomal number was mainly from hypertriploid to hypotetraploid range. The modal number of cells at 33rd passage was 81. Specific marker chromosome is not realized. The production of SCC antigen is detected from the cells and the amount of SCC antigen in cultured media was recorded from 1.5 to 2.0 ng per 1 x 10(4) cells for 48 hours. CEA synthesis is also confirmed immunohistochemically.     

Characterization of an HPV-negative cell line (FR-car) derived from a cervical squamous intraepithelial lesion

Summary A new cell line, FR-car, has been established from a biopsy of a low-grade human cervical squamous intraepithelial lesion (SIL). We confirmed the epithelial origin of the cells by keratin staining using polykeratin, AE1/AE3 and CAM 5.2 antibodies. Sixty percent to 80% of the cultured cells stained positive for proliferative cell nuclear antigen (PCNA) and Ki-67. There was no overexpression of p53. Karyotyping revealed that the cell line was hypodiploid with clonal abnormalities on chromosome 6 and 16. Sections of a biopsy adjacent to the lesion from which the culture was initiated tested positive for human papillomavirus (HPV) 18 DNA by the polymerase chain reaction, but cultured cells tested at several passages were HPV-negative by either type-specific or consensus PCRs. This HPV-negative SIL line may be useful in studies into the cell biology of dysplastic epithelium.     

Simultaneous flow cytometric detection of nuclear DNA and tumor-associated antigens in lung cancers

Flow cytometric (FCM) determinations of DNA index were found to be insufficient to distinguish the presence of tumor cells from normal ones in neoplastic tissues obtained from 29 patients with lung cancer. Therefore, the DNA and tumor-associated antigen (TAA) contents of cultured human lung cancer cells were simultaneously analyzed using FCM to assess whether this dual technique would help in distinguishing tumor cells from normal ones. For the study, cells from PC-10 (a squamous cell carcinoma line), PC-3 (an adenocarcinoma line) and PC-6 (a small cell carcinoma line) were mixed with normal peripheral lymphocytes. The TAAs studied were carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCA) and neuron-specific enolase (NSE). The alcohol-fixed cells were treated with the respective primary TAA, followed by fluorescein-isothiocyanate-conjugated secondary antibody; the cellular DNA was then stained using propidium iodide. Red and green fluorescences were measured simultaneously by FCM. The results showed CEA mainly in PC-3 cells, SCC in PC-10 cells and NSE in PC-6 cells; thus, each cell type had a relatively specific TAA. DNA content and cell size analyses differentiated neoplastic cells from normal lymphocytes for PC-3 and PC-10 cells, but not for PC-6 cells. Simultaneous FCM analyses of DNA and the TAA specific for the individual cell type made it possible to distinguish all tumor cell types from normal lymphocytes.     

Human retinal pigment cell culture

Human retinal pigment epithelium (RPE)-derived cell lines were established from RPE-covered choroid tissue fragments, which had been generated by culture on nontissue culture plastic. Two phenotypes were apparent in a given line: (a) a compact cell which formed domes and ultimately melanosomes before being sloughed; and (b) a squamous cell which was often elongated and which bound antibody to human keratins. This latter cell did not become black or form domes. The average number of cell doublings for the 13 lines tested was between 15 and 40 when cultured in a modified Eagle's minimum essential medium containing 10% fetal bovine serum. Cell lines newly established from material that had been in culture for more than 6 months had normal mitotic chromosomes and still developed areas with strongly pigmented cells when refed. Normal human epithelial cell lines of this kind may be useful in studies of cell aging and defining change associated with the development of neural cells from ectoderm.     

Establishment and characterization of a lymphoepithelial-like carcinoma cell line (HUUCLEC) derived from the human uterine cervix

A cell line designated HUUCLEC was established from a human uterine cervical lymphoepithelial carcinoma obtained from a 61-year-old Japanese woman. The cell line has grown slowly without interruption and serial passages were successively carried out 60 times within 3 years. The cultured cells were spindle or round in shape, showing anaplastic and pleomorphic features, a pavement cell arrangement and multilayering without contact inhibition. The population doubling time of the HUUCLEC line was 72 hours while the chromosomal number varied widely and showed aneuploidy. The modal chromosomal number was stable at the triploid range and marker chromosomes were present; the Ebstein-Barr virus was absent in the cultured cells.     

Stage-related, cell-mediated cytotoxicity with effector cell analysis and computation of cytotoxic activity of T cells and non-T cells.

Cell-mediated cytotoxicity (CMC) by lymphocytes from patients with oral squamous cell carcinoma, as well as from nonmalignant control donors, was tested by a microcytotoxicity assay against a cultured cell line derived from an oral squamous cell carcinoma. In terms of the degree of CMC, stage-related cytotoxicity was observed. A further study of the effector cell analysis revealed that the cytotoxic effects of lymphocytes from both patients and control donors were largely attributable to non-T cells. However, the effectors were also stage related, and in early stage patients, T-cell-mediated cytotoxicity was super-imposed on non-T-cell-mediated cytotoxicity. This conclusion was further supported by the evidence of elevated cytotoxic activity of T cells in early stage patients, which was computed from simultaneous equations proposed in the present paper for computing the cytotoxic activity of T cells (CTA_T) and non-T cells (CTA_non-T).     

人肺癌细胞系HB-99的建立及其生物学特征

利用一例肺鳞癌手术标本通过原代培养建立了肺癌细胞系命名为HB-99。该细胞系呈单层贴壁生长,从相差显微镜和电镜分析具有细胞的多形性,细胞倍增时间为24小时,克隆形成率40%,染色体改变复杂,众数63-65。将细胞移植到裸鼠体内而生长的肿块具有与原始病人手术标本相似的组织形态。免疫组织化学分析,近100%的细胞表达角蛋白17（CK17）,10%的细胞表达波形蛋白（vimentin）。根据该细胞系的生物学特征提示HB-99是一新建立的肺鳞癌细胞系。 Abstract：We have established a human lung squamous carcinoma cell line ,designed HB-99, by culturing primary tumor sample. The cells of HB-99 derived from resected specimen of a male patient with lung squamous cancer. They grew in monolayers and showed cellular morphology by phase contrast and electronic microscopy. The HB-99 cells had a doubling time of 24 hours and a cloning efficiency of 40%. Chromosomal analysis showed complicated rearrangements with a modal number of 63～65. When hetero-transplanted to nude mice, HB-99 grew to form tumor with the same morphology as the original one from the patient. The results of immunohistochemistry suggested that CK17 expressed in almost all cells while only 5%～10% cells had Vimentin. HB-99 is really a newly established cell line of lung squamous carcinoma.     

Serial culturing of human bronchial epithelial cells derived from biopsies

Summary In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.     

Differentiation of normal and tumoral human keratinocytes cultured on dermis: Reconstruction of either normal or tumoral architecture

Summary Normal human keratinocytes isolated from skin and squamous carcinoma cells established from a human tumor (TR146 cell line) both exhibit limited morphologic differentiation when they are grown on conventional plastic dishes. However, when they are seeded on human de-epidermized dermis and cultured at the air-liquid interface, they are able to reform an epithelium having the morphology of the tissue of origin (i.e. skin or squamous carcinoma). The distribution in such reconstructed tissues of differentiation markers such as bullous pemphigoid antigen, 67K keratin, involucrin, membrane-bound transglutaminase, and filaggrin was very similar to their distribution in normal skin and squamous carcinoma specimens, respectively. The degree of differentiation is for both cell types extremely sensitive to culture conditions such as retinoic acid concentration, emersion of the cultures, etc. These results show that subcultured normal or tumoral keratinocytes are able to recover their specific morphogenetic potential when cultured in an environment close to their in vivo situation.     

Transforming Growth Factor β1Induces Squamous Carcinoma Cell Variants with Increased Metastatic Abilities and a Disorganized Cytoskeleton

Previous studies indicated that mouse transformed keratinocytes undergo an epithelial–fibroblastic conversion when cultured in the presence of TGF-β₁. This conversion is associatedin vivowith a squamous-spindle carcinoma transition. We derived epithelioid (A6, FPA6) and spindle (B5) clonal cell variants from a squamous carcinoma cell line (PDV) after treatment with TGF-β₁. FPA6 cells were isolated from the ascites fluid of an A6-tumor-bearing mouse. FPA6 and A6 cell lines produced in nude mice mixed carcinomas with a squamous and poorly differentiated component. Both cell lines coexpressed keratins and vimentin and synthesized E-cadherin protein, although FPA6 cells cultured at early passages (FPA6-ep) had reduced levels of E-cadherin mRNA and increased synthesis of keratin K8, a marker of malignant progression. Immunofluorescence analysis revealed that FPA6-ep cells exhibited a disorganized cytoskeleton with keratins forming focal juxtanuclear aggregates and loss of F-actin stress fibers and cortical bundles, and E-cadherin was localized in the cytoplasm out of cell–cell contact areas. Sporadic cells in A6 and PDV cultures also presented those anomalous keratin structures, suggesting that FPA6 cells originated from a subpopulation of A6 tumor cells that metastasized into the peritoneal cavity. The analysis of the spontaneous and experimental metastatic potentials of the cell lines showed that epithelioid and fibroblastic cell variants had acquired metastatic abilities compared to PDV which was nonmetastatic. The FPA6-ep cell line exhibited a highly aggressive behavior, killing the animals at about 17 days after intravenous injection of the cells into athymic mice. The phenotype of FPA6-ep cells was unstable and reverted at later passages in which the normal organization of keratin and F-actin in filaments and the localization of E-cadherin at cell–cell contacts were restored. This phenotypic reversion occurred concomitantly with a reduction of the experimental metastatic potential of FPA6 cells.     

人胚胎角膜上皮细胞系的建立和生物学特性

为构建角膜基础研究的稳定载体和操作平台,通过合法渠道获得人胚胎,并对角膜上皮细胞进行了分离培养。经反复传代,初步建立了人胚胎角膜上皮细胞系,并对其生物学特性进行了研究。结果显示,培养细胞具有较典型的上皮细胞特征。细胞生长较快,最快时两天可传代一次。K19和PCNA在各代胚胎角膜细胞均有表达,K19的表达部位位于胞浆,而PCNA的表达部位位于胞核。处于细胞周期中S期的细胞比例大约为11%￣23%。染色体核型分析表明各代细胞染色体的数目和形态相似。因此,人胚胎角膜上皮细胞适合于建立相对稳定的细胞系。培养细胞具有分化上的幼稚性和较强且稳定的增殖能力,细胞生长状态良好,而且该细胞系的遗传特征较为稳定。     

Human retinal pigment cell culture

Summary Human retinal pigment epithelium (RPE)-derived cell lines were established from RPE-covered choroid tissue fragments, which had been generated by culture on nontissue culture plastic. Two phenotypes were apparent in a given line: (a) a compact cell which formed domes and ultimately melanosomes before being sloughed; and (b) a squamous cell which was often elongated and which bound antibody to human keratins. This latter cell did not become black or form domes. The average number of cell doublings for the 13 lines tested was between 15 and 40 when cultured in a modified Eagle's minimum essential medium containing 10% fetal bovine serum. Cell lines newly established from material that had been in culture for more than 6 months had normal mitotic chromosomes and still developed areas with strongly pigmented cells when refed. Normal human epithelial cell lines of this kind may be useful in studies of cell aging and defining change associated with the development of neural cells from ectoderm. This work was supported by U.S. Public Health Service Grant AG-00378 to Dr. V. J. Cristofalo.     

Loss of invasiveness in squamous cell carcinoma cells overexpressing desmosomal cadherins

The molecular and structural characteristics of intercellular adhesion were investigated in a squamous cell carcinoma (SCCA) cell line, originally derived from an oral tumor with an invasive growth pattern. The expression of adherens junction and desmosomal components were compared with that of cultured normal oral keratinocytes. Lack of membrane association in interdesmosomal areas, the disorganization of the actin cytoskeleton and the faster cell disassembly upon E-cadherin antibody binding in SCCA cells indicated decreased functional adherens junctions. These observations were supported by a significant reduction in E-, N-, and P-cadherin protein expression. In contrast, the level of desmosomal cadherin proteins, desmoglein 1/2 and desmocollin 2, were substantially upregulated and accompanied, ultrastructurally, by increased number and size of desmosomes. Since tumor invasion suppressor capacity has been proposed for desmosomal cadherins, we investigated the in vivo invasion potential of these SCCA cells by introducing them into SCID mice. Tumors developed, but with a benign phenotype. Based on these results, we hypothesize that the benign behavior of this SCCA cell line is a consequence of overexpressed desmosomal cadherins. This SCCA cell line, therefore, represents an excellent model system to further investigate the regulation and tumor invasion suppressor potential of desmosomal adhesion molecules.     

Notch1 Is a 5-Fluorouracil Resistant and Poor Survival Marker in Human Esophagus Squamous Cell Carcinomas

Notch signaling involves the processes that govern cell proliferation, cell fate decision, cell differentiation and stem cell maintenance. Due to its fundamental role in stem cells, it has been speculated during the recent years that Notch family may have critical functions in cancer stem cells or cancer cells with a stem cell phenotype, therefore playing an important role in the process of oncogenesis. In this study, expression of Notch family in KYSE70, KYSE140 and KYSE450 squamous esophageal cancer cell lines and virus transformed squamous esophageal epithelial cell line Het-1A was examined by quantitative RT-PCR. Compared to the Het-1A cells, higher levels of Nocth1 and Notch3 expression in the cancer cell lines were identified. Due to the finding that NOTCH3 mainly mediates squamous cell differentiation, NOTCH1 expression was further studied in these cell lines. By Western blot analyses, the KYSE70 cell line which derived from a poorly differentiated tumor highly expressed Notch1, and the Notch1 expression in this cell line was hypoxia inducible, while the KYSE450 cell line which derived from a well differentiated tumor was always negative for Notch1, even in hypoxia. Additional studies demonstrated that the KYSE70 cell line was more 5-FU resistant than the KYSE450 cell line and such 5-FU resistance is correlated to Notch1 expression verified by Notch1 knockdown experiments. In clinical samples, Notch1 protein expression was detected in the basal cells of human esophagus epithelia, and its expression in squamous cell carcinomas was significantly associated with higher pathological grade and shorter overall survival. We conclude that Notch1 expression is associated with cell aggressiveness and 5-FU drug resistance in human esophageal squamous cell carcinoma cell lines in vitro and is significantly associated with a poor survival in human esophageal squamous cell carcinomas.     

Integrin- and Cadherin-Mediated Induction of the Matrix Metalloprotease Matrilysin in Cocultures of Malignant Oral Squamous Cell Carcinoma Cells and Dermal Fibroblasts

Matrilysin is a matrix metalloprotease (MMP) overexpressed in a number of cancers including skin, head and neck squamous cell carcinomas, and prostate and colon adenocarcinomas. Matrilysin has been shown to play a role in the degradation of the basement membrane that separates epithelium from stroma allowing tumor cells to intravasate into the bloodstream and metastasize. Here, we show that an oral squamous cell carcinoma cell line (SCC-25) expresses low levels of promatrilysin when cultured alone. However, when SCC-25 cells are cocultured with human foreskin fibroblasts (HFF), there is a 40-fold induction of promatrilysin expression. We tested whether this induction of promatrilysin expression was due to the release of paracrine factors, cell-cell interactions, or cell-matrix interactions. Our results indicate induced promatrilysin expression is the result of both cell-cell and cell-matrix interactions. We demonstrate that beta1 integrins as well as cadherins, specifically N-cadherin and E-cadherin, are involved in the induction of promatrilysin expression. Our results are of general interest in relation to the regulation of MMP expression through cell surface receptor regulation. Further investigation may lead to the identification of novel targets for suppression of invasion and metastasis in oral tumors.     

Identification of Cancer Stem-Like Side Population Cells in Purified Primary Cultured Human Laryngeal Squamous Cell Carcinoma Epithelia

Cancer stem-like side population (SP) cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC) cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs) were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07%) was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC)-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP) LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.     

Establishment and characterization of an immature epithelial cell line (ENU-T-1) derived from a rat nephroblastoma

A new cell line designated ENU-T-1 has been established from a xenotransplanted experimental rat nephroblastoma. The cultured cells are spindle-shaped or polygonal and are arranged in a wavy fashion morphologically similar to cultured embryonal renal epithelial cells. The cells exhibit a number of epithelial characteristics. Enzyme histochemistry gives positive reactions for gammaglutamyltranspeptidase and alkaline phosphatase, both of which are present in renal tubular epithelial cells. Immunofluorescence studies show positive reactions for vimentin and cytokeratin. When inoculated into athymic nude mice, the cultured cells form tumors composed of sheets of epithelial cells with focal tubular formation. This cell line may be of value in studying differentiation of nephroblastoma, and possibly normal nephrogenesis.     

威灵仙多糖对舌鳞癌细胞生长抑制作用的研究

目的:探讨威灵仙多糖对人舌鳞癌细胞Tca-8113的体外生长抑制作用。方法:提取分离纯化威灵仙多糖,用噻唑蓝(MTT)法测定在不同浓度威灵仙多糖作用下人舌鳞癌细胞Tca-8113的活力,观察药物对癌细胞的生长抑制作用。结果:威灵仙多糖对Tca-8113的生长具有明显的抑制作用,随着威灵仙多糖浓度的增大或作用时间延长,抑制作用逐渐增强,呈一定的剂量和时间依赖关系。结论:在体外实验中,威灵仙多糖对人舌鳞癌细胞Tca-8113具有明显的杀伤和抑制作用,可进一步研究其作为抗肿瘤药物应用于临床的潜力。     

Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium containing chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride,culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sulfate at 37℃, 5% CO2. The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to confluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical researches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.