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M F Pera

Publications and source records attributed to M F Pera.

At least 37 records · Page 2Linked to original sources
PubMed1992

A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line.

We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [35S]sulphate resulted in the incorporation of [35S]sulphate into the purified molecule. Throughout the purification procedure, the peaks of 35S radioactivity were coincident with the peaks of immunoreactivity, and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2, indicates that the antigen is not related to previously described keratan sulphate proteoglycans.

Amino Acids↗
PubMed1992

Immunohistochemical and biochemical characterisation of the expression of a human embryonal carcinoma cell proteoglycan antigen in human germ cell tumours and other tissues.

In the embryonal carcinoma (EC) cell line GCT 27, monoclonal antibody GCTM-2 recognises an epitope on a 200 kD pericellular matrix keratan sulphate proteoglycan. Immunohistochemical analyses demonstrated staining of tissue sections from 21 out of 22 human non-seminomatous germ cell tumours, and from 22 out of 28 sections of seminomas. In normal human fetal tissues gut epithelium and muscle stained strongly, and certain other epithelia stained moderately. In adult tissues, the distribution of the epitope was similar, but staining intensity was weaker. Neoplastic tissues showed reactivity with embryonal rhabdomyosarcoma and colorectal carcinoma, but no other non-germ cell tumours. Immunofluorescence microscopy showed that GCTM-2 also stained cell lines from human colorectal carcinoma, embryonal rhabdomyosarcoma and choriocarcinoma. In contrast to EC cells the epitope in these other cell types required permeabilization of the cells to be visualised, and the protein bands in immunoblots lacked extensive modification with keratan sulphate and were smaller. Thus, GCTM-2 reacts with an epitope which has a previously unrecognised tissue distribution; its expression as a pericellular matrix proteoglycan is predominantly a characteristic of human EC cells.

Antibodies, Monoclonal↗
PubMed1991

Monoclonal antibodies to human embryonal carcinoma cells: antigenic relationships of germ cell tumors.

Fifteen monoclonal antibodies (mAb) that show specificity for human embryonal carcinoma cells are described. C57BL/6 mice were immunized with Tera-2 embryonal carcinoma cells, and hybridomas were isolated and tested versus a set of human developmental tumor cell lines. The antigens exhibit relatively restricted and unique distributions on normal tissues as shown by immunohistochemistry. The mAbs recognize a series of differentiation antigens since their expression changes when teratocarcinoma stem cells are induced to differentiate in vitro. The expression of these molecules in germ cell and related tumors is consistent with the data obtained from in vitro cell studies. Seven of these mAbs immunoprecipitate molecules from surface labeled Tera-2 cells that show distinct molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antigenic phenotypes defined by the set of mAbs can be used to investigate possible pathogenetic relationships of the various testicular tumors.

Animals↗
PubMed1990

Comparative biology of mouse and human embryonal carcinoma.

The biology of human germ cell neoplasia has been the subject of increasing investigation during the past decade. The objectives of such investigations include better understanding of the pathogenesis of the diseases, improvement in patient management, and insights into cancer cell differentiation and human development. Many of the approaches to human germ cell tumour biology have been strongly influenced by work in the mouse. Here we review some comparative aspects of germ cell tumours in mouse and man. There are striking differences between the two species in the mechanisms of tumour development, the role of genetic background in tumour susceptibility, the cytogenetics of the tumours, and the phenotype of the embryonal carcinoma stem cells. Despite these differences, certain common themes emerge from a survey of work on growth factors and their receptors, extracellular matrix molecules and transcriptional regulators. Both mouse and human embryonal carcinoma cells produce growth factors, but strong evidence for autocrine regulation of stem cell growth is lacking in both species. Differentiated derivatives (primarily extraembryonic endoderm) of mouse embryonal carcinoma cells may respond to factors produced by the stem cells. These differentiated cells also secrete high levels of growth factors and extracellular matrix molecules in both species, supporting a role for paracrine growth interactions in peri-implantation development. The requirements of embryonal carcinoma stem cells for cell substrate adhesion molecules are specific and differ in mouse and man. There are some similarities in the induction of putative transcriptional regulators by retinoids in human and mouse embryonal carcinoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Isolation and characterization of a multipotent clone of human embryonal carcinoma cells.

Histopathological studies suggest that the stem cells of human teratomas may be classified into two major categories: nullipotent stem cells, and multipotent stem cells, capable both of self-renewal and differentiation into a wide range of somatic and extraembryonic cell types. We have isolated a multipotent stem cell clone from the human teratoma cell line GCT 27, and compared its properties to a nullipotent clone derived from the same strain. The multipotent clone GCT 27 X-1 gave rise to colonies of mixed cell morphology in vitro. Analysis of cell surface, cytostructural and extracellular matrix markers in GCT 27 X-1 cells showed that the stem cells of this line were very similar in phenotype to nullipotent cells. The two cell clones were predominantly hypotriploid, and contained several marker chromosomes in common. GCT 27 X-1 was feeder-cell-dependent for continuous growth in vitro; removal of the feeder layer resulted in differentiation of the stem cells into a variety of cell types, some with characteristics of extraembryonic endoderm, others showing neuronal properties. When transplanted into nude mice, GCT 27 X-1 cells gave rise to teratocarcinomas containing embryonal carcinoma stem cells, and many other cell types: yolk sac carcinoma cells; cells producing alphafetoprotein or human chorionic gonadotrophin; glandular, columnar, cuboidal, and squamous epithelium; primitive mesenchyme and cartilage; neuroectodermal cells. Nullipotent GCT 27 C-1 cells could form colonies in the absence of feeder layers, but multipotent GCT 27 X-1 cells could not. While a range of known growth factors and related substances failed to substitute for feeder layers in supporting the growth of GCT 27 X-1 stem cells, supernatants from yolk sac carcinoma cell line GCT 44 could partially replace the feeder cell requirement. Thus, the results revealed a basic difference in growth control between these multipotent and nullipotent human embryonal carcinoma cells, and suggested a possible paracrine regulatory pathway between multipotent stem cells and yolk sac carcinoma cells.

Animals↗
PubMed1988

Analysis of cell-differentiation lineage in human teratomas using new monoclonal antibodies to cytostructural antigens of embryonal carcinoma cells.

Human embryonal carcinoma cells sometimes display the developmental potential of early embryonic stem cells. While available data do not clearly identify a counterpart of these tumor cells in normal development, previous comparisons of human embryonal carcinoma and yolk sac carcinomas indicated that these cell types are closely related, and suggested that embryonal carcinoma cells might resemble the progenitors of extraembryonic endoderm. To analyse further cell-differentiation lineage in these tumors, we produced monoclonal antibodies to cytostructurally associated antigens of human embryonal carcinoma cells. Spleen cells from mice immunized with a detergent-insoluble extract of cultured human embryonal carcinoma cells were fused to NS-1 myeloma cells, and hybridoma supernatants were screened by indirect immunofluorescence on the immunizing cell line, then on a panel of cell lines derived from human embryonal carcinomas, yolk sac carcinomas, and a range of neoplastic and normal tissues. Monoclonal antibody GCTM-1 stained the nuclei of all human cells tested and served as a positive control; this antibody immunoprecipitated proteins of 85 and 66 k Da from human embryonal carcinoma cells. GCTM-2 recognized an epitope on a 200-k Da extracellular protein present on the surface of embryonal carcinoma cells, and stained the surface of visceral yolk sac-type carcinoma and colorectal carcinoma cells as well. Enzymatic analysis of carbohydrate residues on the GCTM-2 antigen revealed that it was a keratan sulphate proteoglycan, and suggested that the epitope recognized by the antibody lies on the core protein. In immunoblots, antibody GCTM-3 bound to a 57-k Da cytoskeletal protein expressed in human embryonal carcinoma. This antibody decorated filamentous arrays in cell lines from human embryonal carcinoma, visceral yolk sac carcinoma, parietal yolk sac carcinoma (endodermal sinus tumour), and adenocarcinoma and large cell carcinoma of the lung. Antibody GCTM-4 recognized a determinant present on a 69-k Da polypeptide, associated with a component of the lysosomal compartment, which was expressed in embryonal carcinoma cells, but no other cell type tested. The results with this antibody panel thus allow distinction between human embryonal carcinoma and yolk sac carcinoma, but provide further evidence of a close relationship between these cell types.

Animals↗
PubMed1988

Vitronectin production by human yolk sac carcinoma cells resembling parietal endoderm.

Normal mesenchymal cells, normal epithelial cells and many transformed epithelial cells require serum attachment factors and extracellular matrix proteins for growth and differentiation in vitro, and recent evidence strongly supports a role for extracellular matrix molecules in the regulation of cell movement in vivo during early embryogenesis. We previously described the isolation and characterization of cell lines representative of three types of stem cells most commonly found in human adult testicular teratomas, namely embryonal carcinoma cells, yolk sac carcinoma cells resembling visceral endoderm and yolk sac carcinoma cells resembling parietal endoderm (endodermal sinus tumour cells). Of these three cell types, only endodermal sinus tumour cells, which show particularly malignant behaviour in vivo, have no serum requirement for attachment and growth in vitro. Supernatants from endodermal sinus tumour cells support the attachment of embryonal carcinoma cells in serum-free medium. We demonstrate here that endodermal sinus tumour cells, but not other cell types isolated from testicular teratomas, secrete the serum attachment protein, vitronectin (also known as serum-spreading factor, S-protein or epibolin), as well as fibronectin, laminin and type IV collagen, into serum-free medium. Purified vitronectin from medium conditioned by endodermal sinus tumour cells supported both attachment and spreading of embryonal carcinoma cells in vitro, whereas cells attached but did not spread properly on surfaces coated with fibronectin or laminin. Peptides containing the RGD cell recognition sequence common to many attachment proteins blocked attachment of endodermal sinus tumour cells to untreated tissue-culture plastic in serum-free medium. The results suggest a possible role for vitronectin in regulating cell motility and growth in early development, and in the invasion and spread of teratomas in vivo.

Collagen↗
PubMed1987

Inherent sensitivity of cultured human embryonal carcinoma cells to adducts of cis-diamminedichloroplatinum(II) on DNA.

Clinical and experimental data indicate that human embryonal carcinoma cells are unusually sensitive to the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin), but the basis of this sensitivity is unknown. Using colony formation assays we measured survival of cultured human embryonal carcinoma cells following cisplatin treatment and related survival to the amount of platinum bound to DNA, determined by isolation of cellular DNA and flameless atomic absorption spectrometry, over a range of drug doses. Similar measurements were carried out on F9 mouse embryonal carcinoma cells and on a fibroblast cell strain from a patient with the genetic disease Fanconi's anemia, a syndrome associated with hypersensitivity to cytotoxic and clastogenic effects of difunctional DNA-binding agents. These results were compared with similar analyses on a variety of cultured cells from previous studies. The embryonal carcinoma cells and the Fanconi's anemia fibroblast strain were among the most sensitive cells on a dose-response basis. Since the amount of platinum bound to DNA after treatment of these cells was similar to values reported for many other cell types, it follows that mouse and human embryonal carcinoma cells are inherently sensitive to DNA-bound platinum adducts, to a degree approaching the sensitivity of fibroblasts from patients with xeroderma pigmentosum and Fanconi's anemia.

Animals↗
PubMed1987

Cultured stem-cells from human testicular teratomas: the nature of human embryonal carcinoma, and its comparison with two types of yolk-sac carcinoma.

The understanding of differentiation in human teratomas requires a better definition of the phenotype and developmental potential of the stem cells in these tumours. We describe the characterization of 6 new cell lines from human testicular teratomas which are representative of 3 distinct cell types. Cell lines GCT 27, GCT 35, and GCT 48, identified as embryonal carcinoma, comprise epithelial cells which express cytokeratin intermediate filaments and desmoplakins, as determined by indirect immunofluorescence microscopy. A minority of the cells also express vimentin. Most cells in these cultures show surface staining with monoclonal antibodies (MAbs) to stage-specific embryonic antigens SSEA 3 and SSEA 4 but not SSEA 1. Staining with MAb W6/32, which recognizes HLA A,B and C chains in the presence of beta-2 microglobulin, is not above background level. When injected into nude mice, GCT 27 cells form tumours consisting of embryonal carcinoma, somatic tissues, and cells positive in immunocytochemical assays for alphafoetoprotein (AFP) and human chorionic gonadotrophin (HCG); GCT 35 cells form embryonal carcinomas with foci of AFP and HCG-positive cells; and GCT 48 cells form embryonal carcinoma only. A second type of cell (GCT 72) displays some properties of rodent visceral endoderm. GCT 72 cells contain cytokeratin intermediate filaments, but not vimentin, and show very strong staining at cell borders with anti-desmoplakin I + II antibody. At the cell surface, GCT 72 cells express the epitopes recognized by antibodies to SSEA 3, SSEA 4, and SSEA 1; staining with W6/32 is negligible. Levels of AFP in supernatants from GCT 72 cultures are in excess of 500 KIU/I. The tumours formed following inoculation of nude mice with GCT 72 cells are solid yolk-sac tumours, with all cells strongly positive for AFP. A third cell type (GCT 44 and GCT 46), resembles in some ways rodent parietal endoderm. These cells uniformly coexpress keratin and vimentin intermediate filaments, but not desmoplakins. The determinants recognized by MAbs to SSEA 3, 4, or 1 are not detected on the majority of cells in these cultures. In striking contrast to the other teratoma lines, these cells can attach to untreated tissue culture plastic in serum-free medium and may be serially cultivated under these conditions. The tumours formed in nude mice by these 2 cell lines are yolk-sac carcinomas with endodermal sinus tumour histology. Thus, human testicular teratomas consist of epithelial cells which may be nullipotent, pluripotent or committed to extraembryonic endodermal differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

A panel of human lung carcinoma lines: establishment, properties and common characteristics.

A panel of human lung carcinoma lines representing the four main histological types (squamous, small-cell, large-cell and adenocarcinoma), and derived from both primary and metastatic sites, has been established in xenograft and in tissue culture. The highest take rates were achieved when biopsy specimens were obtained from large tumour masses and cultured lines were most readily established after preliminary passages as xenografts. The established lines exhibited an overlapping spectrum of biochemical and morphological characteristics, and showed a tendency to change from one cell type to another, in keeping with the concept of a common endodermal cell of origin. Radiation resistance appeared to be related to the large-cell phenotype.

Adenocarcinoma↗
PubMed1984

In vitro analysis of multistage epidermal carcinogenesis: development of indefinite renewal capacity and reduced growth factor requirements in colony forming keratinocytes precedes malignant transformation.

Using a culture system that supports both extensive proliferation and differentiation of mouse keratinocytes, we analysed alterations in cellular growth control that occur during multistage carcinogenesis in vivo. Normal diploid keratinocytes derived from newborn mouse hair follicles had a limited replicative potential of approximately 50 population doublings in vitro, and colony formation was highly dependent upon cholera toxin, serum growth factors, and mesenchymal feeder cell support. In contrast, keratinocyte lines from benign epidermal lesions or malignant tumors induced by chemicals showed indefinite renewal capacity of the colony forming population, and greatly reduced dependence on cholera toxin and serum growth factors. Cell lines from benign lesions did not readily produce tumors in syngeneic hosts, but cells derived from malignant tumors produced squamous cell carcinomas within a short latent period.

Animals↗
PubMed1984

Differential effects of complete and second-stage tumour promoters in normal but not transformed human and mouse keratinocytes.

The complete tumour promoter phorbol, 12-myristate, 13-acetate (PMA) induces terminal differentiation in the majority of normal cultured human and mouse keratinocytes but a subpopulation exists which is resistant to this effect (PMAR). We have compared with PMA the effects of mezerein (Mez) and phorbol, 12-retinoate, 13-acetate (PRA) on the ability of normal and transformed human and mouse keratinocytes to terminally differentiate in an attempt to elucidate why the latter two compounds are inefficient complete tumour promoters but are effective as second-stage promoters when given after PMA in the two-stage promotion regimen. Both PMA and Mez increased cornified envelope formation in a similar way in normal and transformed keratinocyte cultures inducing a 20- to 25-fold increase over the solvent controls in normal keratinocytes but only a 2-fold increase in line SCC-27 (a cell line derived from a human squamous cell carcinoma). However, while quantitative dose response studies of the effect of phorbol esters on colony forming ability revealed a proportion of normal human and mouse keratinocytes which were resistant to PMA, no normal keratinocytes were resistant to Mez or PRA. In contrast, cell lines derived from papillomas and squamous cell carcinomas showed a resistant fraction of similar size with all three compounds. Furthermore, when Mez or PRA were mixed with PMA the survival of line SCC-27 was the same as when the cultures were treated with the compounds individually indicating that the keratinocytes which were resistant to PRA or Mez were also the PMAR subpopulation. A non-tumorigenic subclone of line SCC-12 (clone F.2), previously shown to possess all known properties of transformed keratinocytes except defective terminal differentiation in suspension culture responded to PMA and Mez in a similar way to normal keratinocytes, suggesting that resistance of the PMAR subpopulation to second-stage promoters requires the expression of a defect in the keratinocyte terminal differentiation programme.

Carcinogens↗
PubMed1982

Toxicity of cisplatin and hydroxymalonatodiammine platinum (II) towards mouse bone marrow and B16 melanoma in relation to DNA binding in vivo.

The antitumour selectivity of cisplatin and hydroxymalonatodiammine platinum II (Pt[OHmal(NH3)2]), a second generation platinum drug, was evaluated in C57B1/Cbi mice bearing advanced intramuscular B16 melanoma. At maximally tolerated doses, Pt[OHmal(NH3)2] produced greater inhibition of growth of the advanced B16 melanoma than did cisplatin. Comparison of dose-response curves for survival of B16 lung colony-forming cells and bone marrow stem cells treated in vivo indicated that selective killing of B16 cells was achieved with Pt[OHmal(NH3)2], whereas cisplatin was relatively nonselective. The extent of reaction of platinum with DNA at doses producing measurable levels of survival in tumour and marrow in vivo was similar to values previously observed in cultured cells treated with cisplatin in vitro. Studies of the amount of platinum bound to DNA in tumour and marrow following administration of the two drugs revealed that the improved selectivity of Pt[OHmal(NH3)2] was associated with a selective increase in the amount of platinum bound to tumour DNA, relative to cisplatin. In addition, platinum lesions produced in DNA by Pt[OHmal(NH3)2] appeared to be more effective in producing tumour cell killing than those produced by cisplatin. No significant excision of total DNA-bound platinum from tumour was observed up to 48 h after administration of either drug.

Animals↗
PubMed1981

Quantitative aspects of the formation and loss of DNA interstrand crosslinks in Chinese hamster cells following treatment with cis-diamminedichloroplatinum(II) (cisplatin). II. Comparison of results from alkaline elution, DNA renaturation and DNA sedimentation studies.

The formation of interstrand crosslinks in the DNA of cis-diamminedichloroplatinum(II) (cisplatin)-treated Chinese hamster cells has been demonstrated by three independent, highly sensitive techniques: namely those of alkaline elution, renaturation of crosslinked DNA and alkaline sucrose gradient velocity sedimentation. Simultaneous measurement of DNA break frequency by the first two methods and a computer model combined with the third enabled the calculation of crosslink frequency after application of low doses of cisplatin. Good agreement was found between the values obtained by the three methods used here, and also with those obtained by direct measurement of the amount of crosslinked hybrid DNA in density-labelled cells treated with higher doses of cis-platin (Roberts, J.J. and Friedlos, F. (1981) Biochim. Biophys, Acta 655, 146--151). When measured by all three methods described here, crosslinking was found to increase during several hours after treatment and then to decrease with a half-life of between 12 and 24 h. For low initial levels of crosslinking, this was largely attributed to an excision repair process, since the formation of breaks in DNA was only minimal.

Animals↗