Involvement of c-Jun in the control of glucocorticoid receptor transcriptional activity during development of chicken retinal tissue.

The ability of the glucocorticoid receptor (GR) to induce gene expression in embryonic chicken retinal tissue increases dramatically during development, although the quantity of the receptor molecules does not change greatly with age. This study examines the possible involvement of c-Jun in the developmental control of GR activity. Expression of c-Jun in retinal tissue was high at early embryonic ages and declined during development. Elevation of c-Jun expression in retina of mid-developmental ages by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or by introduction of a c-Jun expression vector, caused a pronounced decline in the inducibility of the endogenous glutamine synthetase gene and the transiently transfected CAT constructs p delta G46TCO and pGS2.1CAT, that are controlled by a minimal consensus glucocorticoid response element (GRE) promoter and the glutamine synthetase promoter, respectively. The effect of c-Jun was dose dependent and could be reversed by overexpression of GR. C-Jun-evoked repression of GR activity could be relieved by overexpression of Jun D. Overexpression of Jun D could also elevate the responsiveness of early embryonic retina to glucocorticoids and cause a 5-fold increase in p delta G46TCO induction. The effect of Jun D could be reversed by overexpression of c-Jun. Expression of c-Jun might therefore be important for repression of GR activity at early embryonic ages.

In vivo acquisition of Fc gamma RII expression on polyoma virus-transformed cells derived from tumors of long latency.

BALB/c 3T3 cells transformed in vitro with polyoma virus were cloned and passaged once in syngeneic mice. Resulting tumors from each clone were explanted and recultured. Expression of receptor for Fc of IgG (Fc gamma RII) in the original in vitro maintained clones and in cells derived from tumors elicited by the respective cells was measured at the protein level as well as at the mRNA level. Clones were assayed in pairs. The ancestor in vitro maintained clones [designated cultured cells (C)] were compared with cells derived from the same clones after a single passage in vivo followed by explantation and reculturing [designated cultured-tumor-cultured cells (CTC)]. C cells of any of the tested clones did not express Fc gamma RII. On the other hand, certain CTC cells were positive. The Fc gamma RII-positive cells were derived from tumors appearing after a long precancer latency period (greater than 140 days). CTC cells derived from tumors that appeared after shorter latency periods (less than 80 days) were Fc gamma RII negative. These results were obtained both by using radioimmunoassay and monoclonal antibodies against mouse Fc gamma RII as well as by Northern blot analysis using the Fc gamma RII complementary DNA probe. The involvement of macrophages as the Fc gamma RII-expressing cells in CTC cells was excluded. Fc gamma RII expression was down-regulated in CTC cells as a function of time following their explantation into culture. Fc gamma RII expression could be up-regulated in these cells and induced on C cells by maintaining the cultured cells in the presence of normal mouse serum or recombinant interferon. We also tested the expression of Fc gamma RII on CTC cells following their inoculation into syngeneic mice for a second time (CTCx2 cells). The results showed a positive correlation between Fc gamma RII expression in the inoculated ancestor CTC cells and on the CTCx2 cell progeny.

Some cellular and molecular characteristics of high and low tumorigenicity variants of polyoma-virus transformed cells.

We analyzed several cellular and molecular properties of BALB/c 3T3 cellular clones transformed in vitro with polyoma virus and exhibiting a high or low tumorigenicity phenotype. We also analyzed the same clones after a single in vivo passage in syngeneic mice. This passage invariably induced and/or selected variants exhibiting a very high tumorigenicity phenotype. BALB/c mice bearing tumors induced by the inoculation of the above cells, regardless of their tumorigenicity phenotype, have a lower number of L3T4 positive splenocytes than appropriate controls. The response to Con-A of spleen cells from such mice was also suppressed. Concomitantly, an increase in Mac-1 positive splenocytes could be measured. In spite of the non-specific suppression of T cells, spleen cells from tumor-bearers showed a specific proliferative response to polyoma antigens. Molecular analysis of polyoma transformed cells showed no differences between the various cells with respect to integration of the polyoma viral genes or with respect to src, myc and fos proto-oncogenes. In vitro maintained cells and in vivo passaged cells seemed to differ, however, in the content of polyoma middle T. Whereas polyoma virus transformed cells maintained only in culture never expressed low affinity receptors for IgG (Fc gamma RII), certain in vivo passaged cells did. This expression could be measured both at the protein and the mRNA level. Those in vivo passaged cells which expressed F alpha RII gave tumors following a long latency period. Ongoing experiments will indicate whether or not Fc gamma RII expression is linked to long latency of tumor development.

Carcinogen-induced DNA amplification in vitro: overreplication of the simian virus 40 origin region in extracts from carcinogen-treated CO60 cells.

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.