Post-translational processing of membrane-associated recombinant human stem cell factor expressed in Chinese hamster ovary cells.

This report describes the structure of soluble human stem cell factor isolated from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with stem cell factor (SCF) cDNA, which encodes a leader sequence plus 248 additional amino acids. The 248 amino acids include a hydrophobic transmembrane region at positions 190-212. The isolated material is glycosylated and three bands (apparent M(r) 28,000, M(r) 35,000, and M(r) 40,000) are evident by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After complete deglycosylation, the molecular weight by SDS-polyacrylamide gel electrophoresis is 18,000-19,000. Structural analyses of the intact SCF, the deglycosylated SCF, and a deglycosylated C-terminal peptide were performed by laser desorption, fast atom bombardment, or electrospray mass spectrometry. Pulse-labeling of cells with 35S-labeled Met and Cys resulted in cell-associated glycosylated SCF of M(r) 33,000-45,000 which was converted to M(r) 33,000 by in vitro treatment with glycosidases. During a chase with unlabeled Met and Cys, labeled SCF of M(r) 28,000, M(r) 35,000, and M(r) 40,000 appeared in the medium; it was converted to M(r) 18,000-19,000 by glycosidase treatment. SCF at the surface of the transfected CHO cells could be demonstrated by immunofluorescence. The data obtained indicate that the recombinant human stem cell factor, as isolated, represents proteolytically processed forms containing amino acids 1-165, derived from the initially synthesized membrane-bound form of 248 amino acids. Further characterization indicated that the M(r) 28,000 form is glycosylated at Asn120, the M(r) 35,000 form at Asn120 and Asn65, and the M(r) 40,000 form at Asn120, Asn93, and Asn65. Each form also contains O-linked carbohydrate. The N-linked glycosylation, particularly that at Asn93 and at Asn65, adversely affects in vitro biological activity and receptor binding.

A reassessment of methylcholanthrene transformation in the C3H10T1/2 cell culture system.

We previously demonstrated that four tumorigenic methylcholanthrene (MCA) transformed cell lines derived from C3H10T1/2 cells each contain a common G34----T nucleotide alteration in the c-Ki-ras gene. In contrast, a non-tumorigenic MCA transformant does not contain this mutation. We have now examined 75 newly isolated MCA transformants of C3H10T1/2 cells for their degree of morphological transformation, the presence of the c-Ki-ras G34----T mutation, colony formation in soft agar, and tumorigenicity in nude mice. Although many of these new MCA transformants exhibit morphological characteristics indistinguishable from previously isolated tumorigenic MCA transformants, none contain the G34----T mutation in the c-Ki-ras gene. Only one newly isolated MCA transformant can grow in soft agar. Of 14 tested, none of the new MCA C3H10T1/2 transformants are tumorigenic in nude mice.

The tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid differ in toxicity, mitogenic activity and induction of gene expression.

Okadaic acid (OA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are both potent tumor promoters in a mouse skin carcinogenesis experiment. OA was much more toxic than TPA for murine embryo cell lines such as Swiss 3T3 cells or C3H10T1/2 cells. TPA is a potent mitogen for 3T3 cells; in contrast OA was unable to stimulate DNA synthesis in these cells. TPA induces a family of primary response genes, the TPA induced sequence (TIS) genes, in a wide variety of cells. Although OA induced modest levels of TIS mRNA expression, the time course of the induction of TIS1 and TIS8 mRNA was delayed when compared to induction by TPA or peptide mitogens such as fibroblast growth factor (FGF). In addition TPA-mediated down-regulation of protein kinase C attenuated TIS gene induction by OA, but not by FGF.

Colony size, cell density and nature of the tumor promoter are critical variables in expression of a transformed phenotype (focus formation) in co-cultures of UV-TDTx and C3H10T1/2 cells.

UV-TDTx cells are cloned from foci arising after C3H10T1/2 cells are sequentially exposed to u.v. irradiation followed by tetradecanoylphorbol acetate (TPA). When grown in pure culture, UV-TDTx cells appear transformed. Co-culture with C3H10T1/2 cells suppresses focus formation by the UV-TDTx cells. In the presence of TPA, however, focus formation by UV-TDTx cells occurs in C3H10T1/2 co-cultures. We now demonstrate that only tumor promoters that activate protein kinase C (TPA, teleocidin) can reverse C3H10T1/2 suppression of UV-TDTx focus formation in co-culture; other promoters (diethyl stilbestrol, dioxin, saccharin, cadmium) are inactive. Retinoic acid, a potent inhibitor of many biological effects of TPA, blocks the action of TPA in UV-TDTx:C3H10T1/2 co-cultures. Focus formation by UV-TDTx cells in co-culture is dependent on the size of the UV-TDTx colony at confluence; if the UV-TDTx colony is below a minimal size when the co-cultures reach density-dependent growth arrest, suppression of focus formation by C3H10T1/2 cells occurs even in the presence of TPA. Finally, TPA must be present prior to confluence to relieve suppression of focus formation. If TPA is added to co-cultures after density arrest, UV-TDTx cells will not subsequently form foci.

Ultraviolet irradiation transforms C3H10T1/2 cells to a unique, suppressible phenotype.

Transformation of C3H10T1/2 cells by exposure to ultraviolet (UV) irradiation followed by tetradecanoyl phorbol acetate (TPA) has been used as a model of two-stage carcinogenesis. However, cells cloned from UV-TPA-induced foci (UV-TDTx cells) had a unique phenotype. Cloned UV-TDTx cells appeared transformed in pure culture but were unable to form foci when cocultured with C3H10T1/2 cells. However, in the presence of TPA, UV-TDTx cells form foci in mixed culture with C3H10T1/2 cells. This phenotype was the only one observed for UV-TPA transformants. These data suggest that communal suppression of cell division is a discrete phenomenon that must be overcome as one step in the multistage process of transformation, and this protocol permits the routine isolation of transformed cells responsive to density-dependent growth suppression.

Enhancement of oncogenesis in C3H/10T1/2 mouse embryo cell cultures by saccharin.

Impure and pure samples of saccharin (2 milligrams per milliliter) did not produce oncogenic transformation of C3H/10T1/2, clone 8, mouse embryo fibroblasts. However, after treatment of the cells with a nontransforming initiating dose (0.1 microgram per milliliter) of 3-methylcholanthrene, continuous treatment with either sample of saccharin (100 micrograms per milliliter) led to significant transformation. It is concluded that in this system saccharin is a cocarginogen, probably functioning as a promoting agent that is 1000-fold less active than the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate.

Effects of promoters on DNA synthesis in C3H/10T1/2 mouse fibroblasts.

The synthesis of DNA has been studied by autoradiography and by measurements of tritiated thymidine ([3H]TdR) incorporation in cultured C3H/10T1/2 mouse embryo fibroblasts. The cells were first treated with 3-methylcholanthrene as an initiator and then with promoters according to schedules that produce oncogenic transformation. The levels of 3-methylcholanthrene used did not affect the growth or [3H]TdR incorporation of the cells. Treatment during the log phase of growth with 12-O-tetradecanoyl-phorbol-13-acetate, phorbol didecanoate, or 4alpha-phorbol didecanoate produced a transient inhibition of [3H]TdR incorporation with the maximum at 12 hr after treatment. This resulted in a temporary delay of growth followed by recovery of the normal cell-doubling time. Phorbol did not produce these effects, suggesting that the inhibition of DNA synthesis is associated with the process of promotion. Although treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate during stationary phase resulted in a 2- to 3-fold stimulation of [3H]TdR incorporation, multiple treatments spanning log and stationary phases were found to be necessary for promotion.

Two-stage chemical oncogenesis in cultures of C3H/10T1/2 cells.

The initiation and promotion stages of chemical oncogenesis have been demonstrated in cultured C3H/10T1/2 mouse embryo cells. Treatment of the cells with a subeffective concentration of 3-methylcholanthrene, benzo(a)pyrene, or 7,12-dimethylbenz(a)anthracene, followed 4 days later by a nontransforming amount of tetradecanoylphorbol acetate (TPA), phorbol didecanoate, or 4-alpha-phorbol didecanoate, produced transformation. Phorbol was ineffective. TPA did not select for transformed cells. When TPA treatment preceded 3-methylcholanthrene, no enhancement of transformation was observed. When TPA was added immediately after hydrocarbon treatment, there was a significant inhibition of transformation. TPA did not exert promoting activity when the hydrocarbons were in high enough concentrations to produce appreciable transformation. The promoting action of TPA cannot be attributed only to a stimulation of cell division.