transforming-growth-factor-alpha and Teratocarcinoma

We wished to explore the role of transforming growth factor (TGF)-alpha in mouse embryonic development.. We examined the gene expression of TGF-alpha and epidermal growth factor receptor (EGFR) in mouse blastocysts by the reverse transcription-polymerase chain reaction and evaluated the effects of TGF-alpha on the development of preimplantation mouse embryos using TGF-alpha antisense oligodeoxynucleotide. Mouse teratocarcinoma F9 cells were also a subject of this study.. Gene transcripts of TGF-alpha and EGFR were present in both blastocysts and F9 cells. TGF-alpha significantly stimulated the rate of blastocoel expansion in early-cavitating blastocysts and the proliferation of F9 cells. Northern blot analysis showed that TGF-alpha gene expression in F9 cells was markedly suppressed in the presence of TGF-alpha antisense oligodeoxynucleotide. TGF-alpha antisense oligonucleotide significantly reduced the rate of blastocoel expansion and the growth of F9 cells. The inhibitory effects of TGF-alpha antisense oligonucleotide on blastocysts and F9 cells were reversed by the addition of TGF-alpha.. The present observations suggest that TGF-alpha acts as an autocrine factor in the development of preimplantation mouse embryos.

Topics: Animals; Antibodies, Monoclonal; Blastocyst; Blotting, Northern; Chorionic Gonadotropin; Electrophoresis, Agar Gel; ErbB Receptors; Female; Gene Expression Regulation, Developmental; Male; Mice; Oligonucleotides, Antisense; Polymerase Chain Reaction; Pregnancy; RNA, Messenger; Teratocarcinoma; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

The multipotent human teratocarcinoma (TC) cell NTera-2 clone D1 (abbreviated NT2/D1) differentiates into a neuronal lineage after retinoic acid (RA) treatment and a distinct phenotype after hexamethylene bisacetamide (HMBA) treatment. We previously reported that RA treatment of NT2/D1 cells reduces cellular cloning efficiency and nude mouse tumorigenicity. This accompanied a loss of mRNA expression of transforming growth factor-alpha (TGF-alpha) and the fibroblast growth factor kFGF, also known as hst-1 (abbreviated hst-1/kFGF). This study extends prior work by reporting that the distinct phenotype induced by HMBA also decreases cloning efficiency, tumorigenicity, and TGF-alpha and hst-1/kFGF mRNA expression in NT2/D1 cells. These RNA findings were confirmed by measurements of growth factor protein in the conditioned media of inducer-treated and untreated NT2/D1 cells. In two established human TC lines refractory to the actions of RA, N2102ep and Tera-1, RA fails to decrease expression of either growth factor despite induction of its nuclear receptor, RAR-beta. However, HMBA induces morphologic maturation and down-regulation of these growth factors in N2102ep cells. This indicates that the loss of TGF-alpha and hst-1/kFGF expression serves as a new marker of differentiation in human TCs. To explore the effects of these growth factors on growth and differentiation of NT2/D1 cells, TGF-alpha or hst-1/kFGF protein was added following inducer treatment or no treatment. Neither growth factor blocked immunophenotypic differentiation, but both promoted the growth of uninduced NT2/D1 cells in cloning assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetamides; Antineoplastic Agents; Cell Differentiation; Cell Division; Cell Line; Clone Cells; Drug Resistance; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Gene Expression; Humans; Proto-Oncogene Proteins; Teratocarcinoma; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (RA) treatment of the multipotent human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. NT2/D1 cells basally express transforming growth factor alpha (TGF-alpha) mRNA and secreted protein. After RA-treatment TGF-alpha expression is markedly reduced. This decline in TGF-alpha expression accompanies the induction of the neuronal phenotype and a marked reduction of tumorigenicity in athymic mice. This suggested a causal link between reduced TGF-alpha expression and the induced differentiation or loss of tumorigenicity of these RA-treated TC cells. To evaluate this possibility, an RA-refractory NT2/D1 subclone was analysed. This subclone, designated NT2/D1-R1, failed to induce differentiation or to decrease TGF-alpha expression despite RA treatment. To further explore the relationship between TGF-alpha expression and RA actions in this human TC cell, a TGF-alpha cDNA was stably transfected and expressed in NT2/D1 cells. RA-treatment of independently obtained TGF-alpha over-expressing clones and a representative control transfectant only expressing the neomycin resistance gene produced a neuronal phenotype similar to parental NT2/D1 cells as assessed by morphologic, immunophenotypic, and gene expression markers of differentiation. RA-treatment of these clones also induced a G1 arrest similar to parental cells. However, only the TGF-alpha over-expressing clones that secreted high levels of TGF-alpha protein into the conditioned media before and after RA treatment still developed tumors in athymic mice despite prior exposure to these cells to RA. This finding demonstrates that TGF-alpha can inhibit the anti-tumorigenic effects of RA in human TCs. Thus, over-expression of a single growth factor that normally declines with RA treatment antagonizes the anti-tumorigenic but not the differentiation actions of RA in this human tumor cell.

Topics: Animals; Blotting, Northern; Cell Transformation, Neoplastic; Culture Media, Conditioned; DNA, Neoplasm; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Immunophenotyping; Mice; Mice, Nude; RNA, Messenger; Teratocarcinoma; Transfection; Transforming Growth Factor alpha; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured