Transactivation properties of wild-type and mutant androgen receptors in transiently transfected primary human fibroblasts

BACKGROUND: Stromal cells play key roles during androgen-mediated male sexual differentiation. Our objective was to establish a transient transfection method for primary human fibroblasts enabling functional characterization of wild-type (wt) and mutant androgen receptor (AR) plasmid constructs, corresponding to partial and complete androgen insensitivity syndrome (PAIS/CAIS). METHODS: An AR-negative fibroblast strain (ARD842) was established from the gonads of a CAIS patient. Wt-AR or either mutants L712F (PAIS), R774C or V866M (CAIS) were transfected using a polyamine-based procedure. Alternatively, two AR-positive male foreskin fibroblast strains were investigated. Androgen-induced activation of two co-transfected reporter plasmids ((ARE)(2)TATA-, MMTV-luciferase) was measured. RESULTS: All three fibroblast strains showed a ligand-dependent rise of luciferase activity after transfection of wt-AR. Mutant plasmids were assessed in AR-negative ARD842 cells. While L712F showed high partial activity, R774C and V866M were nearly inactive. The intrinsic AR of normal foreskin fibroblasts revealed no measurable ligand-inducible reporter gene activity. CONCLUSIONS: Polyamine-based transfection of AR plasmids into cultured fibroblasts provides a promising tool for analysis of AR transactivation, thereby considering a stromal cellular background. This is supported by the mutant ARs which showed the expected levels of impaired transactivation with respect to the corresponding AIS phenotypes. The role of the intrinsic AR in normal male human foreskin fibroblasts needs further exploration.     

Efficient quiescent infection of normal human diploid fibroblasts with wild-type herpes simplex virus type 1

Quiescent infection of cultured cells with herpes simplex virus type 1 (HSV-1) provides an important, amenable means of studying the molecular mechanics of a nonproductive state that mimics key aspects of in vivo latency. To date, establishing high-multiplicity nonproductive infection of human cells with wild-type HSV-1 has proven challenging. Here, we describe simple culture conditions that established a cell state in normal human diploid fibroblasts that supported efficient quiescent infection using wild-type virus and exhibited many important properties of the in vivo latent state. Despite the efficient production of immediate early (IE) proteins ICP4 and ICP22, the latter remained unprocessed, and viral late gene products were only transiently and inefficiently produced. This low level of virus activity in cultures was rapidly suppressed as the nonproductive state was established. Entry into quiescence was associated with inefficient production of the viral trans-activating protein ICP0, and the accumulation of enlarged nuclear PML structures normally dispersed during productive infection. Lytic replication was rapidly and efficiently restored by exogenous expression of HSV-1 ICP0. These findings are in agreement with previous models in which quiescence was established with HSV mutants disrupted in their expression of IE gene products that included ICP0 and, importantly, provide a means to study cellular mechanisms that repress wild-type viral functions to prevent productive replication. We discuss this model in relation to existing systems and its potential as a simple tool to study the molecular mechanisms of quiescent infection in human cells using wild-type HSV-1.     

Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.     

Effect of cytomegalovirus on cell cycle progression and formation of pathological mitoses in cultured human diploid fibroblasts

The effect of cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G2, or M); at the same time, at least part of infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained 3H-thymidine label above chromosomes. This fact suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of cytomegalovirus.     

Isolation of ouabain-resistant human diploid fibroblasts

Seventeen clones resistant to the cytotoxic action of ouabain were isolated in culture by direct selection from 5 independent strains of diploid human fibroblasts. Resistant clones were recovered at frequencies on the order of 10^?7 per wild type cell selected from populations treated with the mutagen EMS, but no resistant cells were detected among 10⁸ unmutagenized cells. Most selected clones remained ouabain-resistant following further propagation in the absence of drug. The growth of wild type cells was inhibited by 50% at ouabain concentrations of 2–5 × 10^?8 M, while resistant clones required 15–180 fold higher drug concentrations to cause equivalent inhibition. Ouabain-resistant clones showed increased resistance of K+ transport function to ouabain inhibition that paralleled their increased resistance to growth inhibition. Initial experiments suggest that under selective conditions the resistant diploid fibroblasts differ significantly from wild type in binding of ³H-ouabain per unit surface area. The ouabain-resistant cells were similar to wild type in transport properties unrelated to ouabain inhibition. Resistant cells had normal karyotypes and senesced with a lifespan similar to control clones. The ouabain-resistant phenotypes of these diploid human fibroblast isolates apparently reflect point mutations that specifically affect the Na+/K+ transport ATPase with respect to ouabain-binding and/or response to bound ouabain.     

Chromosome replication in aging human diploid fibroblasts

The patterns of termination of DNA replication in human embryonic MRC-5 fibroblasts at four passage levels have been examined by autoradiography. Only chromosome 9 showed statistically significant differences in the time of replication among cultures of different ages. This chromosome terminated replication earlier at later passages than at earlier passages, primarily because of differences in the time of replication of the centromere region. Because very few differences were observed at different passage levels, we conclude that changes in the order of chromosome replication are unlikely to contribute to the phenomenon of in vitro senescence.     

Messenger RNA regulation in human diploid fibroblasts

In resting, non-growing human diploid fibroblasts the amount of rRNA is reduced 1.8-fold, cytoplasmic polysomes are disaggregated, and the level of poly-A RNA (mRNA) is reduced 1.8-fold in relation to growing cells. The distribution of poly-A RNA is altered in resting, non-growing cells so that an average of 64% of the total cytoplasmic poly-A RNA sediments along with particles lighter than 80S (prepolysomal) in sucrose density gradients. By comparison, in growing cells only 30% of the cytoplasmic poly-A RNA sediments in the prepolysomal region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly-A RNA from resting cells resembles that of polysomal poly-A RNA from those cells. In contrast, the average size of prepolysomal poly-A RNA from growing cells is much smaller than that of the polysomal poly-A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly-A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one-quarter to one-third of the prepolysomal poly-A RNA of resting cells is recruited into polysomes in the presence of cycloheximide.     

Mutagenic effect of BUdR in diploid human fibroblasts

It has only recently been possible to demonstrate the expected mutagenic effect of 5-bromodeoxyuridine (BUdR) in heteroploid hamster cells in culture. We have now extended this observation to diploid human fibroblasts utilizing techniques adapted from the work of Albertini and DeMars on X-ray mutagenesis at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in these cells. In four separate experiments, fibroblasts from a female donor were exposed to 500 micrograms/ml ethylmethane sulfonate (EMS) or 3 micrograms/ml BUdR yielding survivals of 9% and 5%, respectively. After a 6-day expression period, survivors were plated in selection medium containing 0.3 micrograms/ml 8-azaguanine (8-AG). After 3-5 weeks, azaguanine-resistant colonies were isolated for characterization or stained for counting. The average spontaneous mutation rate/cell/generation was 0.6.10(-6). The average induced mutation rates for EMS and BUdR were 7.8.10(-6) and 6.3.10(-6)/cell/generation, respectively. Similar results were obtained in two experiments with an additional fibroblast line. Mutant colonies isolated following BUdR treatment demonstrated from 1.4 to 61.5% of the HGPRT activity of the parental line and showed at least 8% Barr bodies, excluding the possibility of contamination by Lesch-Nyhan cells. This demonstration of a BUdR effect comparable to that of an alkylating agent or X-irradiation opens the study of mutation due to base-analog substitution in diploid human cells.     

Interactions of human cytomegalovirus with human fibroblasts

Vonka, Vladimir (Baylor University College of Medicine, Houston, Tex.), and Matilda Benyesh-Melnick. Interactions of human cytomegalovirus with human fibroblasts. J. Bacteriol. 91:213-220. 1966.-Virus attachment of human cytomegalovirus to human embryo lung fibroblasts was found to be temperature-independent, from 4 to 37 C. Prolonged incubation at 4 C, however, resulted in inactivation of a high proportion of attached virus. Virus penetration seemed to be temperature-dependent, occurring at 37 C but not at 4 C. Detailed studies of the growth curve of the virus were made. Cell-associated virus preceded the appearance of virus in the fluid phase by 2 to 5 days. Complement-fixing antigen could be detected, but only when the cytopathic effect was advanced, and it was demonstrable only in the cell-associated fraction. Under methyl cellulose, decreasing the bicarbonate concentration in the overlay from 0.225 to 0.15% resulted in marked increase in plating efficiency with all strains tested. However, varying the concentration of bicarbonate from 0.3 to 0.15% in fluid medium did not influence the growth of virus.     

Gene dosage effects in human diploid and tetraploid fibroblasts

The effects of cell ploidy on the biochemical characteristics of cultured cells were compared using human diploid vs tetraploid fibroblasts isolated with a non-selective method. Their DNA replication was compared by thymidine incorporation, and DNA content by Feulgen staining and quantitative analysis. Their RNA and protein content, cell sizes and the specific activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) were assayed quantitatively. With the exception of RNA content, all other parameters demonstrated a 2-fold increase reflecting the increase in cell ploidy. These direct gene dosage effects on the genetic material and functional expression of the human genome were in contrast to previous observations in other species and validate the use of human intraspecific euploid hybrids for biochemical and genetic studies.     

Retinoids induce sister-chromatid exchanges in human diploid fibroblasts

Vitamin A palmitate, retinoic acid and an aromatic retinoic acid analogue (Ro 10-9359) induced sister-chromatid exchanges (SCE) in human diploid fibroblasts. The same SCE level was reached with the 3 compounds tested, when the concentration of vitamin A palmitate was 10 times higher than those of retinoic acid and Ro 10-9359. No correlation was found when the dose-related SCE induction of a retinoid was compared with the respective antineoplastic activity as reported by others.     

Transport ofl-histidine by human diploid fibroblasts in culture

Summary The transport ofl-histidine has been characterized in skin derived diploid human fibroblasts, cultured under strictly controlled conditions. The transport measurements were made on cells grown to subconfluency after 60 to 90 min timed preincubation. The data, at substrate concentrations ranging from 0.050 to 10 mmol/l, were analyzed by a computer program. A saturable transport system (K _m =0.25 mmol/l, V _max =17 nmol/mg protein per min) and a nonsaturable component of influx (K _d =1.6±0.4 nmol/mg protein/min per mmol) were found.l-Histidine displayed no Na⁺ requirement at either low or high concentrations. Inhibition analysis demonstrated thatl-histidine uptake at low concentration was poorly inhibited by amino acids known to be effective inhibitors of system A. The largest fraction ofl-histidine uptake was inhibited by 2-amino-bicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), leucine, and tryptophan. These results indicated thatl-histidine is transported in human fibroblasts, mainly by the Na⁺ independent system L. The differences between this cell type and others studied previously are discussed. This work was supported in part by Grant 773 from UER de Médecine, Université Paris XI (France).     

Functional characterization of wild-type and mutant human sialin

The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes and several diseases are caused by defective sialic acid metabolism. Sialic acids cleaved off from degraded sialoglycoconjugates are exported from lysosomes by a membrane transporter, named sialin, which is defective in two allelic inherited diseases: infantile sialic acid storage disease (ISSD) and Salla disease. To develop a functional assay of human sialin, we redirected the protein to the plasma membrane by mutating a dileucine-based internalization motif. Cells expressing the plasmalemmal construct accumulated neuraminic acid at acidic pH by a process equivalent to lysosomal efflux. The assay was used to determine how pathogenic mutations affect transport. Interestingly, while two missense mutations and one small, in-frame deletion associated with ISSD abolished transport, the mutation causing Salla disease (R39C) slowed down, but did not stop, the transport cycle, thus explaining why the latter disorder is less severe. Since neurological symptoms predominate in Salla disease, our results suggest that sialin is rate-limiting to specific sialic acid-dependent processes of the nervous system.     

Increased glycolysis in ageing cultured human diploid fibroblasts

With increasing population doubling in vitro, human diploid fibroblasts exhibited a highly significant increase in glucose uptake from the growth medium and a corresponding increase in lactate production. The switch to glycolysis occurred prior to the onset of changes in intracellular glucose and lactate concentrations or in the specific activity of the glycolytic regulatory enzyme, pyruvate kinase, it also preceded the morphological alterations held to be characteristic of cellular senescence.