Showing papers by "Michael B. Sporn published in 1983"

Role of retinoids in differentiation and carcinogenesis.

Abstract: It has been known for more than 50 years that retinoids, the family of molecules comprising both the natural and synthetic analogues of retinol, are potent agents for control of both cellular differentiation and cellular proliferation (70). In their original clas sic paper describing the cellular effects of vitamin A deficiency in the rat, Wolbach and Howe clearly noted that there were distinct effects on both differentiation and proliferation of epithelial cells. During vitamin A deficiency, it was found that proper differentia tion of stem cells into mature epithelial cells failed to occur and that abnormal cellular differentiation, characterized in particular by excessive accumulation of keratin, was a frequent event. Furthermore, it was noted that there was excessive cellular proliferation in many of the deficient epithelia. Although the conclusion that an adequate level of retinoid was necessary for control of normal cellular differentiation and proliferation was clearly stated in the original paper by Wolbach and Howe, a satisfactory explanation of the molecular mechanisms underlying these effects on both differentiation and proliferation still eludes us more than 50 years later. It was inevitable that the basic role of retinoids in control of cell differentiation and proliferation would eventually find practical application in the cancer field, and there have been great ad vances in this area, particularly for prevention of cancer. Many studies have shown that retinoids can suppress the process of carcinogenesis in vivo in experimental animals (for reviews, see Refs. 7, 33, 51, 54, 56, and 57), and these results are now the basis of current attempts to use retinoids for cancer prevention in humans. Furthermore, there is now an extensive literature on the ability of retinoids to suppress the development of the malignant phenotype in vitro (for reviews, see Refs. 6, 8, 30, and 31 ), and these studies corroborate the use of retinoids for cancer prevention. Finally, most recently, it has been shown that reti noids can exert effects on certain fully transformed, invasive, neoplastic cells, leading in certain instances to a suppression of proliferation (30) and in other instances to terminal differentiation of these cells, resulting in a more benign, nonneoplastic pheno type (10,11,60,62). Even though there are many types of tumor cells for which this is not the case (33, 52) (indeed, at present there are only a limited number of instances in which such profound effects of retinoids on differentiation and proliferation of invasive tumor cells have been shown), this finding neverthe less has highly significant implications for the problem of cancer treatment. It emphasizes that in many respects cancer is fun damentally a disease of abnormal cell differentiation (36,44), and it raises the possibility that even invasive disease may eventually be controlled by agents which control cell differentiation rather than kill cells. Since carcinogenesis is essentially a disorder of cell differentiation, the overall scientific problem of the role of retinoids in either differentiation or carcinogenesis is essentially

Polypeptide transforming growth factors isolated from bovine sources and used for wound healing in vivo

Abstract: Transforming growth factors, which are polypeptides that induce the transformed phenotype in nonneoplastic cells, have been isolated in bulk amounts from bovine salivary gland and kidney. In experiments in which wound healing chambers were implanted subcutaneously in the backs of rats, these bovine transforming growth factors accelerated the accumulation of total protein, collagen, and DNA in treated chambers. These studies thus show an effect of an isolated transforming growth factor in vivo.

Purification and initial characterization of a type beta transforming growth factor from human placenta

Abstract: A polypeptide transforming growth factor (TGF) that induces anchorage-dependent rat kidney fibroblasts to grow in soft agar has been isolated from human placenta and purified to homogeneity. This polypeptide is classified as a type beta TGF because it does not compete with epidermal growth factor (EGF) for membrane receptor sites but does require EGF for induction of anchorage-independent growth of indicator cells. Purification of this peptide was achieved by acid/ethanol extraction of the placenta, followed by gel filtration, cation exchange, and HPLC of the acid-soluble proteins. Homogeneity of the TGF-beta from the final column was shown by its constant specific activity and amino acid composition across the peak of soft agar colony-forming activity and by its migration as a single band at Mr 23,000-25,000 on NaDodSO4/polyacrylamide gel electrophoresis. Under reducing conditions, the protein migrated on a gel as a single band at Mr 13,000. The purified placental TGF-beta caused half-maximal growth stimulation of indicator cells in soft agar at 64-72 pg/ml (3 pM) in the presence of EGF at 2 ng/ml (0.34 nM).

Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type alpha and type beta transforming growth factors

Abstract: Sarcoma growth factor (SGF) derived from conditioned medium of Moloney sarcoma virus-transformed cells and partially purified by gel filtration (crude SGF) has been characterized by its ability both to compete with epidermal growth factor (EGF) for binding to membrane receptors and to induce anchorage-independent growth of untransformed cells. We now show that further purification of crude SGF by reverse-phase HPLC on muBondapak C18 and CN columns at pH 2 resolves it into two distinctly different polypeptides, which we call types alpha and beta transforming growth factors (TGFs). Type alpha TGF (TGF-alpha), but not type beta TGF (TGF-beta), competes for binding to the EGF receptor and induces the formation of small colonies (1,000-2,000 micron2) of normal rat kidney cells in soft agar. Both TGF-beta and EGF or TGF-alpha must be present in order to induce the formation of large colonies (7,000-15,000 micron2). Based on EGF competing equivalents as determined from a radioreceptor assay with 125I-labeled EGF in normal rat kidney cells, the relative ability of EGF and TGF-alpha to potentiate TGF-beta-dependent colony formation is in the order conditioned-medium TGF-alpha greater than EGF greater than intracellular TGF-alpha. Suboptimal concentrations of the same polypeptides give additive potentiation of the TGF-beta-dependent colony-forming response; saturating levels potentiate a similar maximum response whether used alone or in various combinations. The data indicate that the EGF-competing activity of crude SGF is due to its TGF-alpha component alone, whereas the soft-agar colony-forming activity is due to the combined action of two distinct polypeptides, TGF-alpha and TGF-beta.

Transforming growth factors from neoplastic and nonneoplastic tissues.

Abstract: Transforming growth factors (TGFs) are a heterogeneous family of polypeptides that induce anchorage-independent growth in nonneoplastic anchorage-dependent cells. They have been found in many tissues, both neoplastic and nonneoplastic. All TGFs isolated thus far are of low molecular weight (6000-25,000), are acid and heat stable, and are inactivated by reagents that reduce disulfide bonds. TGFs have been classified as type alpha or type beta based on their interactions with the receptor for epidermal growth factor (EGF) and their requirement for EGF (or an EGF-like polypeptide) for functional activity. TGF-alpha and TGF-beta act synergistically. TGF-alpha induces phosphorylation of tyrosine in the EGF receptor. TGF-beta, isolated from bovine sources, accelerates experimental wound healing in rats.

Structure-activity relationships of a new series of retinoidal benzoic acid derivatives as measured by induction of differentiation of murine F9 teratocarcinoma cells and human HL-60 promyelocytic leukemia cells.

Abstract: A new group of retinoids, which are 3-alkyl and 3-alkoxy derivatives of ( E )-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]-benzoic acid (TTNPB), has been synthesized. They were tested in vitro for biological activity by measuring induction of terminal differentiation in both murine F9 teratocarcinoma cells and human HL-60 promyelocytic leukemia cells. In the F9 assay, TTNPB was significantly more active (dose effective in achieving a half-maximal response, 3 × 10-9 m) than was all- trans -retinoic acid, while the 3-methyl derivative of TTNPB was not. However, in the HL-60 assay, the 3-methyl derivative of TTNPB was more active (dose effective in achieving a half-maximal response, 2 × 10-8m) than was all- trans -retinoic acid, while TTNPB was less active. 3-Ethyl and 3-propyl derivatives were much less active than was all- trans -retinoic acid in both assays. These new retinoids should be useful agents for experimental studies of both cancer prevention and treatment, as well as for investigation of the mechanism of action of retinoids in the control of cell differentiation.

Retinoids and suppression of carcinogenesis.

Abstract: Retinoids—vitamin A and its analogues—have been recognized for more than half a century to be potent agents in the control of cellular differentiation and proliferation. Only recently, however, have there been concerted efforts to exploit this observation, with particular emphasis on cancer prevention. Experimental findings of tumor inhibition and suppression are reviewed.

Variables in the High-Pressure Cation-Exchange Chromatography of Proteins*

Abstract: The use of cation-exchange high-pressure liquid chromatography for the separation of proteins has been investigated. Several factors, including solvent composition, pH, flow rate, and temperature, were examined for their effects on the resolution of protein standards (insulin, β-lactoglobulin, and carbonic anhydrase B; molecular weight range, 6000 to 30,000 and pI range, 5.3 to 6.5). An initial comparison was made of the recovery of these proteins from three commercially available columns (Whatman Partisil SCX, Separation Industry CM silica, and MCB Reagents Lichrosorb KAT). In general, under the conditions employed, the SCX column gave the highest recovery of applied protein. Based on this recovery data, the Partisil SCX column was chosen for subsequent examination of chromatographic parameters that would optimize protein resolution. An increase in temperature decreased retention and resolution but increased recovery, with some proteins being affected more than others. A decrease in pH in the final eluant or an increase in pH in the initial eluant caused an increase in retention times. For some proteins, the decrease in pH resulted in a greater total recovery of protein. This information has been applied to the purification by cation-exchange high-pressure liquid chromatography of transforming growth factors from a human tumor cell line.

Purification by Reverse-Phase High-Performance Liquid Chromatography of an Epidermal Growth Factor-Dependent Transforming Growth Factor*

Abstract: Transforming growth factors (TGF) are low molecular weight, acid-stable polypeptides that confer a malignant phenotype on nonneoplastic cells. TGF from murine sarcoma virus transformed 3T3 cells were isolated by acid/ethanol extraction, Bio-Gel P-30 chromatography, and reverse-phase high-performance liquid chromatography. Using a μBondapak C 18 column with an acetonitrile gradient, TGF activity can be resolved into two peaks, one of which requires epidermal growth factor (EGF) to induce colony formation of indicator cells in soft agar. Subsequent rechromatography of the EGF-dependent TGF on a μBondapak CN column using an n-propanol gradient resulted in a 430-fold purification over the acid/ethanol extract and showed soft agar activity at a concentration of 4 ng/ml in the presence of 2 ng/ml EGF. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one main band with an apparent molecular weight of 13,000.