Gene Activation in WI-38 Fibroblasts Stimulated to Proliferate: Requirement for Protein Synthesis

Confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by fresh medium containing 30% fetal-calf serum. Up to 80% of the cells are stimulated to divide, with a peak of DNA synthesis between 15 and 21 hr. 1 hr after the change of medium there is a 70% rise in chromatin template activity. Cycloheximide inhibited the increase in chromatin template activity. A requirement for RNA synthesis was investigated by incubating stimulated and unstimulated cells with 10 μg/ml of actinomycin D. In spite of a 95% inhibition of RNA synthesis in whole cells, purified chromatin from stimulated cells showed the usual increase in template activity. These experiments implicate a requirement for protein synthesis in template activation, and imply that the synthesis of this (or these) protein(s) is independent of RNA synthesis and regulated by a purely translational mechanism.     

The type II insulin-like growth factor receptor does not mediate deoxyribonucleic acid synthesis in human fibroblasts

Two insulin-like growth factor (IGF) receptors, the type I and type II IGF receptors, have been described. While substantial evidence indicates that the type I receptor is involved in the regulation of cell division, it is uncertain if the type II receptor also mediates this response. Similarly, the role of the insulin receptor in mediating DNA synthesis remains controversial. To address these questions, we used a monoclonal antibody (alpha IR-3) to specifically inhibit type I IGF receptor activity and examined the effects of this inhibition on IGF- and insulin-stimulated DNA synthesis in human fibroblasts. WI-38 human embryonic lung fibroblasts have both type I and type II IGF receptors, as determined by cross-linking [125I] IGF-I and [125I]IGF-II to monolayers of these cells. In serum-free medium both IGF-I and IGF-II stimulate DNA synthesis in WI-38 fibroblasts, with half-maximal effects occurring at 1.5 +/- 0.3 (+/- SD) and 3.4 +/- 1.4 nM, respectively. At maximally effective concentrations, however, both hormones stimulate DNA synthesis to equal levels. alpha IR-3 binds to the type I, but not the type II, IGF receptor on WI-38 cells. It also inhibits IGF binding to the type I receptor on these cells. alpha IR-3 competitively inhibited both IGF-I- and IGF-II-stimulated DNA synthesis in WI-38 cells, but had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated DNA synthesis. These results indicate that in WI-38 fibroblasts the mitogenic effects of both IGF-I and IGF-II are mediated through the type I receptor and that the type II IGF receptor is not directly involved in this response. To define the role of the insulin receptor in mediating DNA synthesis we compared the effects of alpha IR-3 on insulin-stimulated DNA synthesis in a variety of human cell lines under identical experimental conditions. With WI-38 and HEL, another human embryonic lung fibroblast cell line, alpha IR-3 competitively inhibited the mitogenic effect of insulin. However, in two other fibroblast cell lines (GM498 and HES) and an osteogenic sarcoma cell line (MG63), alpha IR-3 inhibited IGF, but not insulin-stimulated DNA synthesis. These results indicate that human cell lines differ in the receptor type through which insulin stimulates DNA synthesis and that these differences are intrinsic properties of the cell lines and are not artifacts resulting from differences in experimental conditions.     

Endogenous RNA-Directed DNA Polymerase Activity in Uninfected Chicken Embryos

Early chicken embryos that are either positive or negative for group-specific antigens of avian leukosis viruses contained endogenous RNA-directed DNA polymerase activity. This endogenous DNA polymerase activity was not increased after mixture of soluble DNA polymerases isolated from chicken embryos with disrupted chicken embryo cells. The endogenous activity was resistant to treatment with deoxyribonuclease, and the initial rate of DNA synthesis was partially resistant to actinomycin D. In contrast, over 90% of the endogenous polymerase activity was destroyed by ribonuclease in medium with high salt concentration. The DNA product of the endogenous DNA polymerase activity from chicken embryos did not hybridize with RNA of Rous sarcoma virus or reticuloendotheliosis virus, whereas about 40% of this DNA product hybridized with the RNA from the same chicken-cell fraction. Antibody against DNA polymerase of avian myeloblastosis virus did not neutralize the chicken endogenous DNA polymerase activity. These results demonstrate that uninfected chicken embryo cells contain endogenous RNA-directed DNA polymerase activity that is not derived from avian leukosis or reticuloendotheliosis viruses.     

Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization.

We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs.     

GENETIC FUNCTIONS OF THE CHLOROPLAST OF Chlamydomonas reinhardi: EFFECT OF RIFAMPIN ON CHLOROPLAST DNA-DEPENDENT RNA POLYMERASE

The effect of rifampin, an inhibitor of bacterial DNA-dependent RNA polymerase, was studied in Chlamydomonas reinhardi. It was shown, in vivo and in vitro, that chloroplast-located, but not nuclear, DNA-dependent RNA polymerase is inhibited by this drug. The inhibition of chloroplast RNA polymerase results in the inhibition of chloroplast rRNA synthesis, and thus in the loss of chloroplast ribosomes. The ability to carry out photosynthesis is also lost after prolonged heterotrophic growth in the presence of rifampin, but cell division and chloroplast replication are not affected. It is proposed that chloroplast DNA contains information for chloroplast rRNA, but this DNA does not have the information for chloroplast DNA polymerase. Moreover, the DNA polymerase is not synthesized on chloroplast ribosomes.     

Age-dependent alterations of the rate of RNA synthesis in rat brain cell nuclei.

Endogenous Mn2+ and Mg2+ activated RNA polymerase activity was measured in isolated cell nuclei of brain from rats of different age-groups. It was established that the activity of the nucleoplasmic RNA polymerase is maintained at the level of young animals up to an age of 16 months but is decreased after 24 months. The nucleolar RNA polymerase activity decreases already at 16 months and a higher ratio of the Mn2+:Mg2+ activated RNA polymerase activities has been found to be characteristic for the older animals. By means of stepwise sucrose density gradient centrifugation fractions were obtained from the nuclear preparations highly enriched in cell nuclei of neuronal and glial origin respectively. By measuring the activity of the nucleoplasmic and nucleolar RNA polymerases in these fractions it was found that the elevated ratio of the nucleoplasmic to nucleolar RNA polymerase activity at 16 months of age is a characteristic of the neuronal nuclei while the glial nuclei behave by the opposite manner. A parallelism existing between the age-dependent change of the endogenous RNA polymerase activity and that of perichromatic granules of rat brain cortical cells is discussed.     

Regulation of the Nucleolar DNA-Dependent RNA Polymerase by Amino Acids in Ehrlich Ascites Tumor Cells

Experiments were performed to ascertain the degree to which the amount of amino acids might be one of the regulatory factors that control the activity of the nucleolar RNA polymerase. Assays of the enzymatic activity were done with isolated nuclei from cells incubated with low and high concentrations of amino acids. Soon after the cells were exposed to a medium enriched in amino acids, a rapid increase of nucleolar RNA polymerase activity occurred. A similar result was obtained in cells incubated with lower concentrations of amino acids. However, the rate of ribosomal RNA synthesized was regularly much higher in cells incubated in a medium enriched with amino acids than in a medium low in amino acids. Apparently, the amino acids only controlled ribosomal RNA synthesis. Thus, neither maturation, processing, and transport of nuclear precursors into cytoplasmic ribosomal RNA, nor the synthesis of rapidly labeled RNA was affected.     

Role of DNA-Dependent RNA Polymerase III in the Transcription of the tRNA and 5S RNA Genes

Mouse myeloma cells have previously been shown (L. B. Schwartz, V. E. F. Sklar, J. A. Jaehning, R. Weinmann & R. G. Roeder, submitted for publication) to contain two chromatographically distinct forms of RNA polymerase III (designated III(A) and III(B)). The enzymes are unaffected by low alpha-amanitin concentrations which completely inhibit RNA polymerase II, but they exhibit characteristic inhibition curves (identical for III(A) and III(B)) at higher toxin concentrations. RNA polymerase I was unaffected at all alpha-amanitin concentrations tested. Myeloma RNA polymerases II, III(A), and III(B) appear to be inhibited by the same mechanism, since the toxin rapidly blocks chain elongation by each enzyme. The characteristic alpha-amanitin sensitivity of RNA polymerase III has been employed in studies of the function(s) of the class III RNA polymerases.Isolated myeloma nuclei and nucleoli contińue to synthesize RNA via the endogenous RNA polymerases when incubated in vitro. With nuclei, newly synthesized 4S precursor (pre-4S) and 5S RNA species were detected by electrophoretic analysis either of the total nuclear RNA or of the RNA released into the supernatant during incubation. The synthesis of both pre-4S and 5S RNA species was inhibited by alpha-amanitin, but only at high concentrations; and the alpha-amanitin inhibition curves for these RNAs were identical to those obtained for solubilized RNA polymerases III(A) and III(B). In control experiments it was shown that the endogenous RNA polymerase II activity of isolated nuclei was inhibited by alpha-amanitin concentrations similar to those required to inhibit purified enzyme II. However, 40-50% of the endogenous activity of nuclei and 100% of the endogenous activity of purified nucleoli was completely resistant to the high alpha-amanitin concentrations necessary to inhibit the RNA polymerase III activities. These experiments rule out nonspecific inhibitory effects in the endogenous systems.These results unequivocally demonstrate the role of RNA polymerase III (III(A) and/or III(B)) in the synthesis of (pre) 4S RNAs and a 5S RNA species.     

Pairwise loss of mitotic ability by human diploid fibroblasts

Non-transformed human diploid fibroblasts, like WI-38, cannot be maintained as permanent cell lines. With successive divisions in culture, the cells lose the ability to synthesize DNA. This loss may be due to inheritance, to a shared environment, or to interaction among offspring. We studied the pattern of DNA synthesis in WI-38 daughter pairs. A synchronized population in early prophase was seeded in cloning medium containing tritiated thymidine. Radioautographs were made after three days of incubation. We found that the loss of ability to undergo DNA synthesis was correlated between the members of daughter pairs. Daughter pairs in which one but not both of the cells had incorporated the labelled thymidine were significantly less frequent than if the loss of ability to synthesize DNA were to occur at random. The spatial pattern of loss is consistent with the release of a diffusible inhibitor of DNA synthesis by each newly-formed cell. Daughter pairs in which one or both of the cells incorporate thymidine occur with increasing frequency as the inter-nuclear distance between pair members increases. Interaction between daughter cells is responsible in part for the finite lifespan of human diploid fibroblasts.     

Expression of cell cycle-dependent genes in young and senescent WI-38 fibroblasts.

We studied the expression of 11 cell cycle-dependent genes in senescent WI-38 fibroblasts and compared the results to those obtained in WI-38 cells from early passages (young cells). Every gene we examined is expressed in the senescent cells at levels similar to those in the young cells, including two genes maximally expressed at the G1/S phase boundary--genes for thymidine kinase and histone H3. The results clearly show that senescent, noncycling WI-38 cells are not similar to quiescent cells. Rather, such senescent WI-38 cells may be blocked just prior to the onset of DNA synthesis.     

The role of JH III and ecdysterone in the regulation of the left colleterial gland activities in Periplaneta americana during the reproductive cycle

We studied the in vitro effects of JH III and ecdysterone on RNA synthesis and secretory activities of the left colleterial gland (LCG) in mature females of Periplaneta americana during the reproductive cycle. We found that RNA synthesis is a cyclic and highly dynamic process. The major increases in poly(A)- RNA synthesis occurred 8 hr before the peak leucine incorporation into colleterial polypeptides, whereas high poly(A)+ RNA synthesis coincided with high leucine incorporation. Depending on the stage of the cycle JH III exhibited stimulatory or inhibitory effects, or caused no change in RNA synthesis. JH III stimulated RNA synthesis in glands from stages in the reproductive cycle with high endogenous RNA synthesis. The pattern of protein synthesis was not affected by JH III. Ecdysterone lowered RNA synthesis in the LCG and induced secretory activity in LCGs isolated at stages far removed from ovulation. JH III reduced the inhibitory effect of ecdysterone on RNA synthesis and inhibited the ecdysterone-induced secretory activity. The presence of ovarioles isolated at 62 hr of the reproductive cycle (onset of in vivo LCG secretory activity) in the incubation medium inhibited RNA synthesis in 32 hr LCG but induced secretory activity. We suggest that changes in synthetic and secretory activities are coordinated with ovulation and ootheca formation through the interactions between JH III and ovarioles, and ecdysterone may also play a role.     

Synthesis of nuclear proteins during DNA repair synthesis in human diploid fibroblasts damaged with ultraviolet radiation of N-acetoxy-2-acetylaminofluroene.

We have examined the accumulation of newly synthesized nuclear proteins into nuclei during DNA repair synthesis in confluent WI-38 human diploid fibroblasts damaged with ultraviolet radiation or N-acetoxy-2-acetylaminofluroene. In contrast to a marked stimulation of DNA repair synthesis, stimulation of amino acid incorporation into histone polypeptides or into the various molecular weight classes of nonhistone nuclear proteins was not observed. These results suggest that detectable stimulation of newly synthesized nuclear protein incorporation into nuclei does not accompany DNA repair synthesis induced by ultraviolet radiation or a direct acting chemical carcinogen. At least for the special case of repair, DNA synthesis may be uncoupled from histone synthesis.     

Release of somatomedin-like activity by cultured WI-38 human fibroblasts

Confluent cultures of normal diploid WI-38 human embryonic lung fibroblasts released somatomedin (SM)-like activity into their incubation medium during culture in serum-free medium. This postculture medium (conditioned medium) stimulated cell division in these same cultured WI-38 fibroblasts and 35SO4 uptake by hypophysectomized rat cartilage in vitro. The conditioned medium also contained immunoreactive SM (IRSM) activity which yielded parallel dose-response curves to human serum in a RIA for SM. The IRSM activity measured in conditioned medium was not the artifactual result of effects of possible SM-binding proteins or proteolytic enzymes in conditioned medium. These studies suggest that cultured WI-38 fibroblasts produce and release SM-like activity which has SM-like biological activity and is immunoreactive with a basic SM purified from human plasma Cohn fraction and having similarity with SM-C and insulin-like growth factor-I. Human GH appears to stimulate production and release of IRSM activity by these cells.