Putative androgen receptors distinguished in wild-type and testicular-feminized (Tfm) mice.

The reduced level of putative androgen receptor in the mouse mutant, testicular feminization (Tfm), chromatographs on DNA-cellulose differently from the bulk of wild-type receptors. While the elution maximum for extracts of Tfm/Y kidney is in the 180–190 mM NaCl range, wild-type kidney extracts exhibit two maxima of elution at 140–150 mM NaCl and 180–190 mM NaCl, respectively. For hypothalamus-preoptic area, Tfm/Y has one elution maximum at approximately 180 mM NaCl, while the wild-type exhibits a major elution maximum at 140–150 mM NaCl, with a minor peak at approximately 180 mM NaCl. Mixing experiments between wild-type and Tfm/Y cytosols reveal that the different characteristic elution patterns are intrinsic to the binding complexes and are not conveyed simply by other soluble factors. The distinctive pattern for Tfm indicates that the mutation does not cause merely a reduced level of wild-type receptor. Rather the residual receptor of the mutant may be either an abnormal protein or a minor form of wild-type receptor, not readily seen in wild-type tissue due to the presence of more preponderant species. Differences in the elution profiles of androgen receptor species of wild-type kidney with the two bound androgens, testosterone and dihydrotestosterone, are also presented. A model of the androgen receptor system is proposed which includes several binding classes for androgen ligands and metabolites. In light of aromatization of androgens to estrogens and its probable role in some androgenic responses, we include the “estrogen receptor” in this mechanism.     

Metabolic cooperation in aggregates of embryonal carcinoma cells

Metabolic cooperation may be associated with the processes of compaction and subsequent differentiation in aggregates of embryonal carcinoma cells (ECC). To determine if the gap junctions present in loose and compacted aggregates of H6 ECC are active in metabolic cooperation, aggregates of each type containing a mixture of 5-bromodeoxyuridine- and 6-thioguanine-resistant H6 cells were exposed to HAT medium, 6-thioguanine, or [³H]thymidine. These three methods indicated that some crossfeeding occurred through the small clusters of gap junctions in loose aggregates and more crossfeeding occurred through the larger clusters of gap junctions in compacted aggregates.     

Neocarzinostatin induction of DNA repair synthesis in HeLa cells and isolated nuclei.

The antitumor antibiotic neocarzinostatin that causes DNA strand breaks in vivo and in vitro is shown to induce DNA repair synthesis in HeLa S3 cells. In the repair assay, the parental DNA was prelabeled with 32P and a density label (bromodeoxyuridine) was introduced into the new synthesized DNA. Quantitation of the repair synthesis as measured by the incorporation of [3H]thymidine into the light parental DNA at varying doses of the drug indicate that there is a significant repair response at low levels of the drug (0.2--0.5 microgram/ml) which cause DNA strand breakage and inhibition of DNA synthesis. In isolated HeLa nuclei neocarzinostatin stimulates the incorporation of dTMP many-fold. This enhancement of dTMP incorporation, which requires the presence of a sulfhydryl agent, is a consequence of the drug-induced DNA strand breakage and is in the parental DNA. These results suggest that an intact cell membrane is not required for DNA strand breakage and its subsequent repair.     

Correlation between synthesis and methylation of DNA in HeLa cells

For the whole cell cycle the methylation of DNA was studied in synchronized $\text{HeLa}$ cells and in nuclei isolated from them. In the intact cells the methylation of DNA cytosine runs parallel to DNA synthesis. The pattern of DNA cytosine methylation by the isolated nuclei is almost identical to that obtained with the whole cells. Since the isolated nuclei do not synthesize DNA, it is shown that DNA methylation continues for at least 30 min after DNA synthesis is over. No DNA minor thymine is found in the isolated nuclei.     

Metabolic cooperation between vascular endothelial cells and smooth muscle cells in co-culture: changes in low density lipoprotein metabolism

A microcarrier co-culture system for aortic endothelial cells and smooth muscle cells (SMCs) was developed as a model for metabolic interactions between cells of the vessel wall. Low density lipoprotein (LDL) metabolism in SMCs was significantly influenced by co-culture with endothelium. The numbers of high affinity receptors for LDL was increased more than twofold (range, 2.1-5.6), with concomitant increases in LDL receptor-mediated endocytosis and degradation. These effects reached a plateau at an endothelial cell/SMC ratio of 1. Kinetic analysis of the endocytic pathway for LDL in SMCs indicated that, in co-culture with endothelium, there was no alteration in the binding affinity of LDL to its receptors but that the internalization rate constant declined and the rate constant for degradation increased. This analysis suggested that the formation and migration of endocytic vesicles was the rate-limiting step of enhanced LDL metabolism under co-culture conditions. Two mechanisms by which endothelial cells influenced smooth muscle LDL metabolism were identified. First, mitogen(s) derived from endothelial cells stimulated entry of SMCs into the growth cycle, and the changes in LDL metabolism occurred as a consequence of G1-S transition. Second, SMC lipoprotein metabolism was stimulated in the absence of mitogens by a low molecular weight (less than 3,500) factor or factors. Co-culture was a required condition for the latter effect, suggesting that the mediator(s) may be unstable or that cell-cell communication was necessary for expression. These results (a) demonstrate that vascular cell interactions can modify LDL metabolism in SMCs, (b) provide some insights into the mechanisms responsible, and (c) identify co-culture as an experimental approach appropriate to certain aspects of vascular cell biology.     

Liver DNA synthesis after phenytoin injection in adult NMRI mice

Stimulation of hepatocyte DNA synthesis was observed in adult mice, as measured by the number of parenchymal cells in S phase, at two different times after a single i.p. injection of diphenylhydantoin (DPH): 18 and 25 hr. No such stimulation was obtained for Kupffer or endothelial cells.     

Discrepancies between precursor uptake and DNA synthesis in mammalian cells

The incorporation of tritiated thymidine and deoxycytidine into DNA of x-irradiated mammalian cells was studied. Both inhibition and stimulation were found due to pool changes rather than to effects on DNA synthesis, indicating that precursor uptake can be a misleading method to measure DNA synthesis rate.     

Histone protein and DNA synthesis in HeLa cells after thermal shock

Exposure of suspension-cultured HeLa cells to a 45° thermal shock resulted in cell inactivation and inhibition of both protein and DNA synthesis. DNA synthesis was inhibited in a biphasic manner with a more sensitive (D₀ = 7 min) and a less sensitive (D₀ = 20 min) phase. The less sensitive process was demonstrated to be DNA chain elongation. Transport of thymidine into intracellular pools was significantly less sensitive to thermal shock (D₀ in excess of 200 min). When HeLa cells were heated at 45° for 15 min there was an 80% inhibition of incorporation of precursors into both DNA and protein with little effect on precursor transport into cellular pools. While the rate of synthesis of whole cell and histone protein (H2a, H2b, H3, and H4) and DNA chain elongation recovered by 6 h after cell heating, total precursor incorporation into DNA was only 0.4 of control levels. The long-term depression of the DNA synthetic rate could not be explained by a cell cycle redistribution, a depression in the total fraction of S phase cells synthesizing DNA, or by a depression in the rate of DNA chain elongation. We conclude that thermal shock results in a long-term depression in the fraction of cell replicons involved in DNA replication.     

Nm23-H1 is responsible for SUMO-2-involved DNA synthesis induction after X-ray irradiation in human cells

Human cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis activity after X-ray irradiation which is suggested to be casually related to reduction in cellular amounts of small ubiquitin-like protein modifier (SUMO-2/SMT-3A). In the present study, an increased level of DNA synthesis activity was found 8 h after X-ray irradiation in HeLa cells with reduction in SUMO-2 amounts by siRNA treatment for SUMO-2. When comparative proteomic analysis was performed between the siRNA and mimic control siRNA treated cells using two-dimensional (2D) electrophoresis and mass spectrometry, three proteins were identified as candidates. Our research focused on Nm23-H1, a nucleoside diphosphate kinase, whose amounts decreased after X-ray irradiation in HeLa cells treated with siRNA for SUMO-2. In the Nm23-H1 siRNA treated cells, induction of DNA synthesis was also detected. Furthermore, in synchronized HeLa cells, DNA synthesis was confirmed in the S phase. Moreover, increased expression of proliferating cell nuclear antigen (PCNA) was observed in Nm23-H1 siRNA treated HeLa cells after X-ray irradiation. In addition, Nm23-H1 was modified with SUMO-2 after X-ray irradiation. The present findings suggest that the reduction of Nm23-H1 is related to the decrease in sumoylation, which in turn, is involved in the induction of DNA synthesis via the regulation of PCNA expression after X-ray irradiation.     

DNA vaccination can break immunological tolerance to PrP in wild-type mice and attenuates prion disease after intracerebral challenge

Transmissible spongiform encephalopathies (TSEs) can be ameliorated by prion protein (PrP)-specific antibodies, but active immunization is complicated by immune tolerance to the normal cellular host protein (PrP(C)). Here, we show that DNA immunization of wild-type mice can break immune tolerance against the prion protein, resulting in the induction of PrP-specific antibody and T-cell responses. PrP immunogenicity was increased by fusion to the lysosomal targeting signal from LIMPII (lysosomal integral membrane protein type II). Although mice immunized with a PrP-LIMPII DNA vaccine showed a dramatic delay in the onset of early disease signs after intracerebral challenge, immunization against PrP also had some deleterious effects. These results clearly confirm the feasibility of using active immunization to protect against TSEs and, in the absence of effective treatments, indicate a suitable alternative for combating the spread of these diseases.     

Relationship between histidyl-tRNA level and protein synthesis rate in wild-type and mutant Chinese hamster ovary cells.

A preliminary investigation was carried out to determine how conditional lethal mutants affected in particular aminoacyl-tRNA synthetases may be used to study the role of tRNA charging levels in protein synthesis. The relationship between rate of protein synthesis and level of histidyl-tRNA in wild-type cultured Chinese hamster ovary cells was determined using the analogue histidinol to inhibit histidyl-tRNA synthetase activity. This response was compared with that obtained using a mutant strain with a defective histidyl-tRNA synthetase that phenotypically shows decreased rates of protein synthesis at reduced concentrations of histidine in the growth medium. The approach used was based on measuring the histidyl-tRNA levels in live cells. The percentage charging was estimated by comparing [14C]histidine incorporated into alkali-labile material in paired samples, one of which was treated with cycloheximide, five minutes before terminating during the incubation, to produce maximal aminoacylation. Wild-type cells under histidinol inhibition exhibited a sensitive, sigmoidal relationship between the level of histidyl-tRNA and the rate of protein synthesis. A decrease in the relative percentage of acylated tRNA (His) from 46% to 35% elicited a large reduction in the rate of protein synthesis from 90% to 30% relative to untreated cells. An unpredicted result was that the relationship between protein synthesis and histidyl-tRNA in the mutant was essentially linear. High acylation values for tRNA (His) were associated with rates of protein synthesis that were not nearly as high as in wild-type cells. These findings suggest that the charging charging levels of tRNA (His) isoacceptors could play a regulatory role in determining the rate of protein synthesis under conditions of histidine starvation in normal cells. The mutant appears to be a potentially useful system for studying the pivotal role of tRNA charging in protein synthesis, assuming that the altered response in the mutant is caused by its altered synthetase.     

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro

Summary Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion of connective tissue cells during the development of liver fibrosis.     

Over-replication of DNA in S phase Chinese hamster ovary cells after DNA synthesis inhibition

Agents that inhibit DNA synthesis increase the frequency of methotrexate resistance and gene amplification in cultured mammalian cells. Chinese hamster ovary cells blocked with hydroxyurea rereplicated dihydrofolate reductase gene sequences within a single cell cycle upon release from the block (Mariani, B.D., and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901-1910). Perturbation of DNA synthesis was postulated to result in misfiring of replicon initiation, subsequent over-replication of DNA sequences, and amplification of specific genes. To test this hypothesis, we have exposed Chinese hamster ovary cells pulsed with bromodeoxyuridine to three agents that inhibit DNA synthesis and enhance gene amplification: UV irradiation, hydroxyurea, and aphidicolin. After release from the block, the progression of cells throughout the cell cycle was analyzed by flow cytometry through simultaneous measurement of total cellular DNA content and bromodeoxyuridine-labeled DNA. Although the cell cycle effects varied depending on the agent used for the block, in all cases a subset of cells that were in S phase at the time of the block exhibited DNA histograms with greater than 4C DNA content at various times after release and prior to cell division. Cells with the excess DNA were approximately 10-fold more resistant to methotrexate compared to treated cells with normal DNA content or untreated cells. Therefore, cells in S phase at the time of the block produce excess DNA per cell prior to division, and this over-replicated DNA may be relevant to gene amplification and drug resistance.