Rat C6 glioma cells express insulin-like growth factor I (IGF-I) and form rapidly growing tumors in syngeneic animals. When transfected with an episome-based vector encoding antisense IGF-I complementary DNA, these cells lost tumorigenicity. Subcutaneous injection of IGF-I antisense-transfected C6 cells into rats prevented formation of both subcutaneous tumors and brain tumors induced by nontransfected C6 cells. The antisense-transfected cells also caused regression of established brain glioblastomas when injected at a point distal to the tumor. These antitumor effects result from a glioma-specific immune response involving CD8+ lymphocytes. Antisense blocking of IGF-I expression may reverse a phenotype that allows C6 glioma cells to evade the immune system.

Malignant glioma is the most common brain tumor. The molecular basis of glioma tumorigenicity has not been defined. Cultured glioma cells accumulate hg levels of insulin-like growth factor I (IGF-I) transcripts. We asked whether IGF-I expression is coupled to tumorigenicity, using a combined in vivo/in vitro system employing antisense RNA for IGF-I. An antisense IGF-I expression construct in an expression vector that incorporates Epstein-Barr virus replicative signals and the ZnSO4-inducible metallothionein I t scriptional promoter was assembled. Stable glioma transfectants were derived from C6 glioma cells, which constitutively express IGF-I. B-104 neuroblastoma cells, derived originally from the same tumor but not expressing IGF-I, were also trasected as controls. In the absence of ZnSO4, the C6 traectants expressed high levels of IGF-I mRNA and protein as detected by in situ hybridization and immunocyochemlsry, respectively. Addition ofZnSO4 in the culture medium resulted in hig levels of antisense transcript accumulation and dramatically de

Teratocarcinoma is a germ-line carcinoma giving rise to an embryoid tumor with structures derived from the three embryonic layers: mesoderm, endoderm, and ectoderm. Teratocarcinoma is widely used as an in vitro model system to study regulation of cell determination and differentiation during mammalian embryogenesis. Murine embryonic carcinoma (EC) PCC3 cells express insulin-like growth factor I (IGF-I) and its receptor, while all derivative tumor structures express IGF-I and IGF-II and their receptors. Therefore the system lends itself to dissect the role of these two growth factors during EC differentiation. With an episomal antisense strategy, we defme a role for IGF-I in tumorigenicity and evasion of immune surveillance. Antisense IGF-I EC transfectants are shown to elicit a curative anti-tumor immune response with tumor regression at distal sites. In contrast, IGF-II is shown to drive determination and differentiation in EC cells. Since IGF-I and IGF-U bind to type I receptor and antisense sequence used for IGF-II cannot form duplex with endogenous IGF-I tran-

The levels of several RNA transcripts in cultured hepatocytes are regulated by transcriptional and posttranscriptional mechanisms and are affected by growth hormone and insulin. We assessed the effects of these hormones on transcription rates and the stability of insulin-like growth factor I, actin, and albumin transcripts in intact cells of primary cultures of rat hepatocytes by analyzing thiol-labeled, newly synthesized RNA isolated by mercurated agarose affinity chromatography. The application of this concept to the measurement of transcript stability is presented in detail. The data indicate that growth hormone stimulates the transcription rates of insulin-like growth factor I, actin, and albumin genes. The stability of all three transcripts, particularly albumin, appears to be lower in growth hormone-containing medium than it is in insulin-containing medium. The experiments indicate that the rates of transcription and/or degradation of albumin mRNA are influenced by hormonal treatment. However, the cells maintain roughly constant albumin transcript levels independent of hormone treatment by compensatory changes in the rates of transcription and degradation.We reported previously (1) that growth hormone (GH) and insulin stimulated the accumulation of insulin-like growth factor I (IGF-I) and actin transcripts in rat hepatocyte primary cultures. By analyzing thiol-labeled, newly synthesized RNA isolated by affinity chromatography with mercurated agarose, we were able to separate the newly synthesized RNA and showed that insulin and GH exerted their effects on IGF-I transcript levels by stimulating transcription. Since it is known that a related hormone, prolactin, has profound effects on the casein mRNA transcript half-life as well as on casein transcription (2), we wished to determine whether GH also affected IGF-I transcript half-life. However, when traditional methodology employing actinomycin D was applied to primary hepatocyte cultures, it was observed that IGF-I transcript levels actually increased in the presence of inhibitor concentrations sufficient to suppress [3H]uridine incorporation by 98%. Similar results have been reported for IGF-I transcripts in U937 cells (3) and rat C6 glial cells (4). This phenomenon was also observed for albumin but not for f3-actin RNA transcripts. Thus, the approach of using transcriptional inhibitors to estimate transcript half-lives is inappropriate for this particular system. In addition, IGF-I transcript levels are not sufficiently abundant to make pulsechase studies readily feasible.In this report we show that analysis of thiolated, newly synthesized RNA, in addition to yielding data on relative transcription rates for specific transcripts under different hormone treatments, can provide estimates of transcript half-life, which are not subject to the same limitations as studies employing transcriptional inhibitors or pulse-chase methods. Moreover, our results indicate that GH increases transcription of actin, albumin, and IGF-I. MATERIALS AND METHODSThe prepa...