This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.

/pdf/isolation-of-a-pluripotent-cell-line-from-early-mouse-2dgu2as4rf.pdf

Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells

https://www.cell.com/cell/pdf/0092-8674(82)90400-7.pdf

The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells

Genistein, a specific inhibitor of tyrosine-specific protein kinases.

During the course of purifying nerve growth factor from the submaxillary gland of the mouse, Cohen (1960) and Levi-Montalcini and Cohen (1960) noticed that daily injections of certain gland extract fractions into newborn mice produced developmental changes that could not be ascribed to nerve growth factor. These changes included precocious opening of the eyelids (7 days compared to the usual 14 days) and a similar early eruption of the incisors. Using these gross anatomical changes as an assay, Cohen (1962) proceeded to isolate the active factor — a polypeptide which he termed epidermal growth factor (EGF).

Epidermal growth factor

The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precursor, deduced from cDNA clones derived from placental and A431 carcinoma cells, reveals close similarity between the entire predicted v-erb-B mRNA oncogene product and the receptor transmembrane and cytoplasmic domains. A single transmembrane region of 23 amino acids separates the extracellular EGF binding and cytoplasmic domains. The receptor gene is amplified and apparently rearranged in A431 cells, generating a truncated 2.8-kilobase mRNA which encodes only the extracellular EGF binding domain.

Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells

Murine sarcoma virus-transformed mouse fibroblasts produce polypeptide growth factors and release them into serum-free medium. These factors stimulate cells to divide in monolayer cultures and also to form colonies that grow progressively soft agar. Three major peaks of activity are seen, with apparent molecular weights of 25,000, 12,000, and 7000. The sarcoma growth factors are heat-stable, trypsin-sensitive, and active in nanogram quantities when tested for growth stimulation of untransformed rat and mouse fibroblasts. All three molecular species are also capable of competing for membrane epidermal growth factor (EGF) receptors when tested with 125I-labeled EGF. They differ from mouse EGF, however, in their molecular weights, in their inability to react with anti-EGF antibodies, and in their ability to convert cells to anchorage independent (agar) growth. For the above reasons, we conclude that the sarcoma growth factors are a new class of polypeptide tropic factors that confer on fibroblasts in vitro properties associated with the transformed phenotype.

https://www.pnas.org/content/pnas/75/8/4001.full.pdf

Growth factors from murine sarcoma virus-transformed cells.

Three different human tumor lines in culture, a rhabdomyosarcoma, a bronchogenic carcinoma and a metastatic melanoma, release proteins (transforming growth factors, TGFs) into the medium that confer the transformed phenotype on untransformed fibroblasts. These proteins are acid and heat-stable; produce profound morphologic changes in rat and human fibroblasts; and enable normal anchorage-dependent cells to grow in agar. Removal of the transforming protein results in a reversion of cell phenotype. The major activity interacts with epidermal growth factor (EGF) cell membrane receptors. The peptides from these tumor cells are similar in their action to the sarcoma growth factor (SGF) released by murine sarcoma virus-transformed rodent cells. The most anchorage-independent tumor cells released the most TGFs. EGF-related TGFs were not detectable in fluids from cultures of cells with high numbers of free EGF membrane receptors (normal human fibroblasts and human carcinomas).

https://www.pnas.org/content/pnas/77/9/5258.full.pdf

Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors

Purified mouse nerve growth factor (NGF)radiolabeled with 125I was tested for its ability to bind to a variety of different cultured cells NGF binds readily to human and hamster melanoma cells but does not bind to many other cell lines The three cell lines with the highest number of NGF receptors were derived from metastatic melanomas One of these lines, A875, was studied in detail and was shown to have approximately 7x10(5) NGF receptors per cell with an association constant of 10x10(9) liters/mole The use of these cells in competition binding assays permits the detection of 025 ng of NGF in various biologic fluids NGF can be shown to increase the survival, but not the division, of melanoma cells maintained in medium depleted of serum growth factors This effect of NGF as a specific "survival factor" appears analogous to its effect on cultured sympathetic ganglion cells and on other cells derived from the neural crest

https://www.pnas.org/content/pnas/74/2/565.full.pdf

Nerve growth factor receptors on human melanoma cells in culture

Polypeptides characterized by their ability to confer a transformed phenotype on an untransformed indicator cell have been isolated directly from tumor cells growing both in culture and in the animal, by using an acid/ethanol extraction procedure. Assay of these polypeptides is based on their ability to induce normal rat kidney fibroblasts to form colonies in soft agar. Peptides from murine sarcoma virus-transformed mouse 3T3 cells grown in culture had the highest specific activity in this assay; peptides from sarcomas produced from these cells or from chemically induced transplantable bladder carcinomas of mice were one-third as active; and peptides from a chemically induced rat tracheal carcinoma had only one-tenth the activity. Treatment with either trypsin or dithiothreitol destroyed the activity of all of these materials. The properties of these intracellular polypeptides from both virally and chemically transformed cells are similar to those described for the sarcoma growth factors (SGFs) previously isolated from the conditioned medium of sarcoma virus-transformed mouse 3T3 cells, suggesting the definition of a class of transforming growth factors common to tumor cells of different origins. The transforming peptides from the cultured sarcoma virus-infected cells were separately by gel filtration into two fractions of apparent molecular weight 7000 and 10,000. The major fraction at molecular weight 7000 represented approximately 0.1% of the original cell protein and had a specific activity 50 times that of the original acid/ethanol extract.

https://www.pnas.org/content/pnas/77/6/3494.full.pdf

Transforming growth factors: isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction

Transforming growth factors (TGFs) were purified from serum-free medium conditioned by retrovirus-transformed Fisher rat embryo fibroblasts, mouse 3T3 cells, and two human melanoma cell lines. The purification of each TGF was monitored in a radioreceptor assay based on receptor crossreactivity with mouse submaxillary gland epidermal growth factor (mEGF) and was achieved by gel permeation chromatography of the acid-soluble TGF-containing activity, followed by reverse-phase high-pressure liquid chromatography with sequential use of acetonitrile and 1-propanol in the presence of aqueous trifluoroacetic acid. The amino-terminal sequences of rat, mouse, and human TGFs were determined. Extensive sequence homology was found among TGF polypeptides from different species and cell types. Alignment of the amino acid sequences of rat TGF, mEGF, and human urogastrone (hEGF) reveals statistically significant sequence homology. The reported results suggest that TGFs that compete for binding to the cellular EGF receptor and EGF may have evolved from a common progenitor.

https://www.pnas.org/content/pnas/80/15/4684.full.pdf

Transforming growth factors produced by retrovirus-transformed rodent fibroblasts and human melanoma cells: amino acid sequence homology with epidermal growth factor.

J E De Larco

Papers

Growth factors from murine sarcoma virus-transformed cells.

Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors

Nerve growth factor receptors on human melanoma cells in culture

Transforming growth factors: isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction

Transforming growth factors produced by retrovirus-transformed rodent fibroblasts and human melanoma cells: amino acid sequence homology with epidermal growth factor.