Flow sorting in the study of cell-cell interaction

Undifferentiated mouse teratocarcinoma cells were cocultivated with differentiated mouse endoderm cells in order to study the possible induction of teratocarcinoma cell differentiation. A difference in DNA content between the two cell types was experimentally introduced to enable the reisolation of the teratocarcinoma cells after cocultivation. Pseudotetraploid (2s) endoderm cell lines were produced from pseudodiploid (1s) cells by treatment of these cells with cytochalasin B and flow sorting of tetraploid cells, using Hoechst 33342 as a viable DNA stain, with subsequent cloning of sorted single cells. In model experiments, where mixtures of 1s teratocarcinoma and 2s endoderm cells were stained with Hoechst 33342, the teratocarcinoma cells could be reisolated with a purity of about 97%. After a cocultivation period of 24 days viable teratocarcinoma cells could be isolated from the cocultivation mixture with a purity of 95%. Two dimensional analysis of the protein pattern of these cells indicated that cocultivation did not induce a differentiated (endoderm) pattern. Therefore according to this analysis the teratocarcinoma cells were not induced to differentiate during a 24 day cocultivation period. The method described offers excellent possibilities for studying cell-cell interaction in vitro.     

Immunohistochemical localization of c-mos at the light and electron microscope level in non-small cell lung carcinomas

C-mos is a cytoplasmic upstream activator of the mitogen-activating protein kinase pathway with serine-threonine kinase activity. It plays a well established and vital role in oocyte maturation by participating in metaphase II arrest and meiotic asymmetric division, but little is known about its function in somatic cells. Recently, we observed overexpressed c-mos in a portion of non-small cell lung carcinomas (NSCLCS). In particular, c-mos immunoreactivity was detected in tumor cell nuclei in addition to its expected cytoplasmic localization, and c-mos overexpression was associated with chromosomal instability among other findings. To verify our earlier observations and to clarify further the role of c-mos in NSCLCS, we examined its distribution by both light and electron microscopy. We detected c-mos in the cytoplasm and/or nucleus of a portion of tumor cells and fibroblasts. In particular, granular immunoreactivity was observed in the cytoplasm closely associated with the rough endoplasmic reticulum. Nuclear staining was confirmed and was often found near the nuclear membrane, as well as in some large multilobular, possibly aneuploid, nuclei. C-mos positivity was also found in the nuclei of tumor cells undergoing apoptosis. Furthermore, c-mos was detected in areas with diminished vascularization. It should be noted that nuclear staining was found at the ultrastructural level more extensively than at the light microscope study. This suggests a masking effect by the hematoxylin nuclear counterstain.     

Supermelanotic hybrids between mouse teratocarcinoma and mouse melanoma cells

Each of four kinds of teratocarcinoma cells, OTT6050P, PCC4, PSA1 and LT, derived from 129 or LT mouse strain, was fused with B16-CAPr melanoma cells derived from C57BL/6J by using Sendai virus. The resultant hybrids were morphologically melanotic melanoma cells which were larger and more heavily pigmented than the parental B16-CAPr melanoma cells. The chromosome analysis and GPI electrophoresis demonstrated that all hybrids were products of fusion between a single teratocarcinoma cell and a single melanoma cell. The pigmentation in the hybrids between a 129 teratocarcinoma cell and a melanoma cell was much stronger than that in hybrids between an LT teratocarcinoma cell and a melanoma cell. This phenomenon was consistent with the difference of coat color between 129 and LT mouse strain. From these results, it was suggested that the genes of teratocarcinoma cells involved in the pigmentation are activated in the hybrids with B16-CAPr melanoma cells.     

Selection of proliferating cybrid cells by dual laser flow sorting : Isolation of teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids

An alternative method for the isolation of proliferating cybrid cells was developed, and was used to obtain teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids. Enucleated neuroblastoma (or endoderm) cells labelled with fluorescent microspheres were fused with (HPRT-deficient) unlabelled teratocarcinoma cells. The cells in the fusion mixture were stained with the vital DNA stain Hoechst 33342 and the cybrids, containing both a fluorescent nucleus and fluorescent beads, were isolated by dual laser flow sorting. The purity of the sorted fraction, as determined by the percentage of cells showing HPRT activity, was 86 and 57% for the neuroblastoma and endoderm cybrids, respectively. After single cell sorting in wells of Terasaki microtest plates, clones of proliferating cybrids were obtained with cloning efficiencies of 33% (neuroblastoma and endoderm cybrids respectively. After single cell sorting in wells of Terasaki microtest parental cell lines were analysed by two-dimensional gel electrophoresis. A number of differences were found between the parental cell lines but all isolated colonies (sixteen neuroblastoma cybrids and eight endoderm cybrids) resembled the teratocarcinoma parent. These results therefore give no evidence for the existence of cytoplasmic factors in neuroblastoma or endoderm cells capable of inducing permanent differentiation of teratocarcinoma cells.     

Calcium-dependent cell-cell adhesion molecules common to hepatocytes and teratocarcinoma stem cells

The molecules involved in Ca2+-dependent cell-cell adhesion systems (CDS) in mouse hepatocytes were characterized and compared with those in teratocarcinoma cells. Fab fragments of antibody raised against liver tissues (anti-liver) inhibited Ca2+-dependent aggregation of both liver and teratocarcinoma cells. A monoclonal antibody raised against teratocarcinoma CDS (ECCD-1) also inhibited the Ca2+-dependent aggregation of these two cell types equally. These antibodies induced disruption of cell-cell adhesion in monolayers of hepatocytes. Thus, CDS in these two cell types are not immunologically distinctive. Immunochemical analyses with these antibodies showed that CDS in both hepatocytes and teratocarcinoma cells involved at least two classes of cell surface proteins with molecular weights of 124,000 and 104,000. ECCD-1 selectively bound to hepatocytes but not to fibroblastic cells in liver cell cultures. Thus, the molecular constitution of CDS in hepatocytes and teratocarcinoma stem cells is identical. As ECCD-1 reacts with other classes of embryonic and fetal cells, the molecules identified here could have a major role in cell-cell adhesion in various tissues at any developmental stage of animals.     

Phenotype and surface antigens of mouse teratocarcinoma x fibroblast cell hybrids

Numerous colonies of hybrids between PCC4-aza 1 teratocarcinoma cells and fibroblasts of the heteroploid Cl.1D cell line were examined. All of the hybrids were fibroblasts showing extinction of the multiple developmental potentialities of the teratocarcinoma cell parent, irrespective of whether the teratocarcinoma parent was diploid or tetraploid. The hybrids did not show loss of any specific chromosome contributed by the PCC4-aza 1 cell parent. In contrast with the PCC4 parental cells which carry F9 antigens and do not express H-2b, the hybrids do not express F9 antigens and carry H-2 alloantigens of both parental specificities. These results suggest that in hybrids whose phenotype is that of the Cl.1D parent, a change may occur in the genetic program of the teratocarcinoma cells.     

Sialic acid lyase in differentiating murine teratocarcinoma cells

N-Acetylneuraminic acid lyase (NAN-lyase) activity has been found to be much higher in the differentiated, murine parietal endodermal cell (PYS-2) in culture than in the related, undifferentiated embryonal teratocarcinoma cell (F9). The level of the enzyme rapidly increases in F9 cells exposed to an inducer of differentiation such as retinoic acid (RA) (10(-7) M). The level of the enzyme increases during log phase of growth of PYS-2 cells and decreases after the cells reach confluence. NAN-lyase from PYS-2 cells has been purified 365-fold and has been partially characterized. While most of the enzyme is freely soluble, at least 16% of the enzyme in PYS-2 cells is associated with the nucleus. The possible function of NAN-lyase in the cell and the significance of its marked elevation during growth and differentiation are discussed in view of the fact that the levels of NAN, neuraminidase, NAN transferases and the enzymes that synthesize and activate NAN, remain essentially unchanged during differentiation.     

Teratocarcinoma cell MHC antigen expression is regulated in vitro by a soluble noninterferon factor

The 402AX murine teratocarcinoma is a spontaneous testicular tumor of 129 (H-2b) origin which does not express MHC encoded antigens. Rejection of this tumor is immunologically mediated and the tumor cells are induced in vivo to synthesize H-2b antigens when passaged in genetically resistant host mice. The present studies demonstrate that serum from tumor primed genetically resistant host mice can induce tumor cell MHC antigen expression in vitro as measured by indirect immunofluorescence using monoclonal antibodies. The inducing factor is specific for 402AX tumor cells and is not interferon as shown by the lack of response of the 402AX tumor to gamma interferon, and the absence of significant interferon activity in inducer serum. These studies demonstrate another factor independent of interferon that can induce MHC class I antigen expression on tumor cells.     

Induction of c-fos and AFP expression in a differentiating teratocarcinoma cell line

The introduction of a c-fos expression vector has been shown to potentiate spontaneous differentiation in teratocarcinoma cells. We have studied a teratocarcinoma stem cell line which can be induced to differentiate with dimethylsulfoxide (DMSO) to determine endogenous c-fos expression during the process of differentiation. c-Fos expression increases dramatically as P19S1801A1 embryonal carcinoma cells are induced to differentiate into a variety of cell types. Expression peaks 12 days after the start of aggregate culture about the same time as alphafetoprotein (AFP), a characteristic of visceral endoderm differentiation, as demonstrated by RNA hybridization to specific probes, ELISA, and immunofluorescent staining with specific antibodies. However, most differentiated cells expressed c-fos, while AFP was expressed in a minor fraction (less than 5%). The data suggest that c-fos is correlated with differentiation of teratocarcinoma cells but not specifically to visceral endoderm formation.     

Establishment of mouse teratocarcinomas stem cells line and screening genes responsible for malignancy

The sequential transplantation of embryonal carcinoma cells in vivo can accelerate the growth and malignancy of teratocarcinomas. However, the possible molecular mechanisms in this process that reflect cancer formation in the early stage are largely unknown and. To identify which genes are associated with the changes of malignancy of teratocarcinomas, we established a tumorigenesis model in which teratocarcinoma were induced via injecting embryonic stem cells into immuno-deficiency mice, isolating teratocarcinoma stem cell from a teratocarcinoma in serum-free culture medium and injecting teratocarcinoma stem cells into immune-deficient mice continuously. By using high-throughput deep sequence technology, we identified 26 differentially expressed genes related to the changes of characteristics of teratocarcinoma stem cell in which 18 out of 26 genes were down-regulated and 8 genes were up-regulated. Among these genes, several tumor-related genes such as Gata3, Arnt and Tdgf1, epigenetic associated genes such as PHC1 and Uty were identified. Pathway enrichment analysis result revealed that Wnt signaling pathway, primary immunodeficiency pathway, antigen processing and presentation pathway and allograft rejection pathway were involved in the teratocarcinoma tumorigenesis (corrected p value<0.05). In summary, our study established a tumorigenesis model and proposed some candidate genes and signaling pathways that may play a key role in the early stage of cancer occurrence.     

Molecularly cloned c-mos(rat) is biologically active.

A unique rat cellular gene, c-mos(rat), homologous to the transforming sequences, v-mos, of Moloney murine sarcoma virus (M-MSV) was detected by hybridization to a v-mos specific probe. The c-mos(rat) gene was cloned together with its flanking sequences in an 11-kbp EcoRI DNA fragment inserted in vector Charon 4A. Two probes were used to investigate the position and orientation of c-mos(rat) in the clone examined ( D3e ), namely pMSV -31 which contains the sequences specific for the transforming sequences of M-MSV and pCS-1 which harbors 0.5 kbp of 5'-terminal sequences of c-mos(mouse) as well as 0.7 kbp of its flanking sequences. After ligation of a restriction fragment of clone D3e containing c-mos(rat) to a fragment containing the long terminal repeat of M-MSV and transfection of the DNA onto rat cells, we detected foci of transformed cells, thus showing that c-mos(rat) is biologically active. Using DNA framents derived from clone D3e , we studied the conservation of c-mos and of its flanking sequences in several species. c-mos(rat) as well as some of its flanking sequences appeared to be highly conserved in the species studied.     

Definitive expression of c-mos in late meiotic prophase leads to phosphorylation of a 34 kda protein in cultured rat spermatocytes

To investigate the role of c-mos in rat spermatogenesis, expression of c-mos, MAP kinase kinase (MAPKK), MAP kinase (MAPK), cdc2 and protein kinase A (PKA) by spermatogenic cell culture of 14 day-old rats was examined. MAPKK and PKA expressions were constitutive, whereas the expression of MAPK and cdc2 in spermatogonia initially decreased, but later increased on meiotic maturation of spermatocytes. c-mos expression was definitive of late meiotic prophase. c-mos immunoprecipitates prepared from the c-mos-enriched fraction (pI9.0-9.6) could form complex(es) in the cultured spermatogenic cell lysates. In vitro phosphorylation of the c-mos immune complexes revealed a 34 kDa protein that was phosphorylated at serine and threonine residues as a target of the c-mos signal. Its pI value was 4.4-4.5, and cdc2 was not detected, making it different from cdc2 (p34). These results suggest that the phosphorylation of the 34 kDa protein by the c-mos signal may play a crucial role in the meiotic division of rat spermatocytes.     

Squamous Cell Differentiation in a Clonal Teratocarcinoma Cell Line

Several clonal teratocarcinoma cell lines restricted to squamous cell differentiation have been established from a subculture of totipotential murine teratocarcinoma stem cells. These lines contain a continuum of cell types from less differentiated precursor cells to differentiated squamous cells. In contrast to their highly malignant progenitors, these cells are nontumorigenic. Chromosomally, the cells are near-tetraploid, and their DNA distributions are tetraploid. These cell lines may prove useful in the study of normal squamous cell differentiation and also will be important in studies concerning the conversion of malignant cells to non-malignant progeny.