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

The effects of several organic and inorganic nitrogen compounds on nitrogenase mRNA and enzyme activity levels were examined in anaerobic cultures ofAnabaena variabiis 29413. Even low concentrations of exogenous ammonia (20 ,uM) Nitrogen-fixing microorganisms synthesize nitrogenase and the other proteins required for nitrogen fixation only under conditions in which nitrogen fixation is required to support growth. In the presence of ammonia and certain other fixed nitrogen compounds, the genetic capacity to fix nitrogen is not expressed. The effects of a variety of inorganic and organic nitrogen compounds such as amino acids and amino acid analogs on nitrogenase synthesis and activity have been studied in several nitrogen-fixing bacteria and cyanobacteria. Overall, the results of many studies show that other utilizable nitrogen sources are usually assimilated in preference to N2, and in the presence of many such compounds nitrogenase activity is absent or greatly reduced (1,10,27). In cyanobacteria, formation of heterocysts is also repressed by compounds which repress nitrogenase synthesis (10, 27). In most of the studies referred to above, investigators have measured nitrogenase activity (using the acetylene reduction technique), so that effects on nif gene regulation were not measured directly. In Klebsiella pneumoniae (3, 5) and Anabaena sp. strain 7120 (11), DNA-RNA hybridization techniques have been used to show that ammonia represses nif gene mRNA levels.As described in the preceding paper (12), we have defined conditions for studying derepression of nitrogenase genes of Anabaena variabilis under anaerobic conditions. This permits rapid experiments to be performed without the complication of heterocyst development. As we describe in this paper, we have used this anaerobic system to study the effects of various nitrogen compounds on nitrogenase gene expression at both transcription and enzyme activity levels. Our results support the conclusion that nitrogenous compounds influence nitrogenase levels primarily at the transcription level, probably by repressing transcription initiation, although some compounds may also have indirect effects on enzyme activity. Also, in agreement with most other studies that have not measured mRNA levels, we find that ammonia itself does not appear to be the immediate effector of nif gene expression. MATERIALS AND METHODSMedia and growth conditions. A. variabilis ATCC 29413 was used for all studies. Cultures were grown as described previously (14).Derepression experiments. Preparation of anaerobic cultures and incubation conditions for derepression experiments were as described previously (12). For experiments with NH4C1, KNO3, glutamine, and glutamate, a series of serum vial cultures was prepared containing cells in media (AA/8 with 0.5% fructose and 10 ,uM dichlorophenyldimethylurea) plus desired concentrations of the test compound; each experiment included a control vial containing no addition. The compound being tested was present throughout the experiment.Because carbamyl...

Gene clones encoding phycocyanin and allophycocyanin were isolated from an Anabaena variabilis ATCC 29413-Charon 30 library by using the phycocyanin (cpc) genes of Agmenellum quadruplicatum and the allophycocyanin (apc) genes of Cyanophora paradoxa as heterologous probes. The A. variabiis cpcA and cpcB genes occur together in the genome, as do the apcA and apcB genes; the two sets of genes are not closely linked, however. The cpc and apc genes appear to be present in only one copy per genome. DNA-RNA hybridization analysis showed that expression of the cpc and apc genes is greatly decreased during nitrogen starvation; within 1 h no cpc or apc mRNA could be detected. The source of nitrogen for growth did not influence expression of the genes; vegetative cells from nitrogen-fixing and ammonia-grown cultures had approximately the same levels of cpc and apc mRNAs. Heterocysts had less than 5% as much cpc mRNA as vegetative cells from nitrogen-fixing cultures. Northern hybridization (RNA blot) analysis showed that the cpc genes are transcribed to give an abundant 1.4-kilobase (kb) RNA as well as two less prominent 3.8-and 2.6-kb species. The apc genes gave rise to two transcripts, a 1.4-kb predominant RNA and a minor 1.75-kb form.

Nonresponsiveness to the growth-stimulatory actions of insulin-like growth factor (IGF)-I in chondrocytes has been reported in a number of disease states associated with impaired glucose metabolism. Primary rabbit chondrocytes were investigated for changes in their IGF response system [type-I IGF receptor and IGF-binding protein (IGFBP) expression] and in their ability to mount a synthetic response to IGF-I [as35S-labeled proteoglycan ([35S]PG) production] in media containing varying ambient glucose concentrations. Whereas basal [35S]PG synthetic rate was unaffected by glucose concentration, synthetic responsiveness to IGF-I was lost in media containing <5 mmol/l glucose or in media containing a “diabetic” glucose concentration (25 mmol/l). IGFBP expression, as measured by Northern analysis of mRNA levels and Western ligand blotting of secreted protein levels, was not significantly altered in the different glucose media, nor were there any differences in the cell surface localization of IGFBPs as assessed by affinity cross-linking with 125I-labeled IGF-I, suggesting that IGFBPs do not induce the IGF-I resistance. The nonresponsiveness to IGF-I in reduced glucose occurred with 25–50% reductions in steady-state levels of IGF type-I receptor mRNA and protein. A significant correlation between IGF receptor mRNA level and synthetic response to IGF-I was observed between 0 and 10 mmol/l glucose concentrations, suggesting that the loss of responsiveness in reduced glucose is manifested at the level of transcription and/or receptor mRNA stability. In contrast, nonresponsiveness to IGF-I in chondrocytes in diabetic glucose concentrations occurred without changes in receptor mRNA and protein levels, suggesting that IGF-I resistance was due to post-ligand-binding receptor defects. It is proposed that IGF-I resistance in chondrocytes subjected to inappropriate glucose levels may constitute an important pathogenic mechanism in degenerative cartilage disorders.