Clive L. Bunn

Decreased accuracy of protein synthesis in extracts from aging human diploid fibroblasts

The accuracy of protein synthesis has been measured in extracts from human diploid fibroblasts of different ages. Extracts were supplied with purified mRNA for the coat protein of the cowpea variant of tobacco mosaic virus (CcTMV), which lacks codons for cysteine and methionine. The presence of 35S-cysteine in CcTMV coat protein synthesized during translation reactions therefore represents translational error. Translation reactions were performed with extracts from young fibroblasts (less than 50% of life span completed) and old fibroblasts (more than 90% of life span completed), and the translation products were purified by immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. The error frequency increased from 4.2 x 10(-5) cysteines/amino acid in young cell extracts to 2.9 x 10(-4) cysteines/amino acid in old cell extracts. Cysteine incorporation was not due to nonspecific binding, and could be increased approximately sixfold by the addition of the misreading antibiotic, paromomycin. It is concluded that translational accuracy is not stable during aging in vitro, and it is proposed that this decrease in the fidelity of information transfer could be responsible for the variety of changes observed in aging cultured human cells.

Metallothionein expression and stress responses in aging human diploid fibroblasts

Metallothioneins (MTs) are low molecular weight proteins with a high cysteine content that are inducible by heavy metals and by other conditions of environmental stress. This laboratory was investigated in human diploid fibroblasts the induction of MTs by cadmium and by dexamethasone, and the induction of heat shock proteins, as models for age-related changes in gene expression that reflect the ability of old cells to respond to environmental stress. Old cells were more sensitive to the toxic effects of CdCl2 in the concentration range 100-175 microM. Analysis of 35S-cysteine-labelled cell extracts by polyacrylamide gel electrophoresis and fluorography showed that in the absence of any inducer, old cells have a 3.7-fold increase over young cells in the basal level of MT. The rate and extent of induction of MT by CdCl2 was reduced in old cells: Exposure of old cells to 100 microM CdCl2 for 18 h resulted in MT levels about 33% of the amount in young cells. Northern blot analysis showed that the changes in MT protein levels occurred in parallel with changes in mRNA levels, which implicates transcriptional control as the origin of these aging changes. These young/old differences in MT synthesis were maintained in density-arrested cultures, indicating that the aging changes were not due to differences in the cell cycle status of these cell populations. The rate and extent of induction of a 68-kDa heat shock protein were also reduced in old cells, which showed an increase in basal, uninduced level of this protein similar to MT. In contrast, old cells retained the ability to synthesize MTs in response to dexamethasone at a rate similar to that in young cells.

Restriction enzyme analysis of mitochondrial DNA in aging human cells

Human diploid fibroblasts show a limited lifespan in vitro. To investigate the integrity of mitochondrial DNA (mtDNA) in aging fibroblasts, whole cell DNA samples from the human cell line IMR-90 have been prepared at 36, 22, and 3 population doublings (PD) from the end of the lifespan (63 PD). These DNA samples were then digested separately with 19 different restriction endonucleases, and the resulting fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose filters. Fragment sizes were revealed by hybridization to 32P-labelled mouse mtDNA and autoradiography, and were compared with computer maps of fragments generated from the known sequence of human mtDNA. These 19 enzymes recognize a total of 297 recognition sites comprising 1315 nucleotide base pairs (bp), approximately 8% of the human mtDNA (16 569 bp). Control experiments reveal that a minor component representing as little as 5% of the total mtDNA can be detected. No changes were seen in the restriction fragment pattern with fibroblast cell age. It is concluded that there are no large deletions, insertions, or rearrangements in human mtDNA, and no single base changes in the detectable regions. This suggests efficient maintenance of mtDNA molecules and/or elimination of damaged mtDNA during fibroblast cell lifespan.

Segregation of mitochondrial DNA in human somatic cell hybrids

SummaryThe maintenance of mtDNA has been examined in human intraspecific hybrid cells constructed from the fusion of HEB7A, a HeLa tumor cell line carrying the mitochondrially coded chloramphenical (CAP) resistance mutation, and GM 2291, a limited lifespan human diploid fibroblast which is CAP sensitive. These two cells can be distinguished by a polymorphism in a site for the restriction endonuclease, HaeIII. Independently isolated clones of hybrid cells were characterized for their growth properties (either normal limited lifespan or transformed and “immortal”). Whole cell DNA preparations were made from each hybrid, digested with HaeIII, and the resultant fragments were detected by hybridization to 32P labelled mouse mtDNA as probe. Experiments with mixtures of HEB7A and GM2291 DNA reveal that HEB7A mtDNA can be detected when it constitutes as little as 5% of the total cell mtDNA.The results indicate that the HEB7A mtDNA is lost from most hybrids, and when it does persist it is usually a minor component of total mtDNA. The addition of CAP at the time of fusion slightly increases the quantity of HEB7A mtDNA, but not enough to confer CAP resistance. Furthermore, five limited lifespan hybrids contained no detectable HEB7A mtDNA, while three transformed hybrids contained varying quantities of HEB7A mtDNA, suggesting that retention of this tumor form of mtDNA is associated with tumor growth behavior. These results suggest that cytoplasmic genetic incompatibility occurs in intraspecific hybrids.

Altered sensitivity of protein synthesis to paromomycin in extracts from aging human diploid fibroblasts

Age-related differences in the effects of paromomycin (Pm) on protein synthesis have been investigated in translation reactions with extracts derived from young and old human diploid fibroblasts. Translation products from reactions directed by endogenous or exogenous mRNA were analyzed by polyacrylamide gel electrophoresis and fluorography. The exogenous mRNA lacked codons for cysteine, and therefore cysteine incorporation into translation products represented translational error. This laboratory has previously used this assay to show that the basal translational error level in the absence of Pm increases in extracts from old fibroblasts. In this report, Pm stimulated the misincorporation of cysteine by 6-7 fold over cysteine misincorporation levels in the absence of Pm. This degree of Pm stimulation was similar in extracts from young and old fibroblasts. However, other results showed quantitative differences in the responses to Pm between young and old cell extracts. Old cell extracts were less sensitive to the stimulation of the rate of protein synthesis, and more sensitive to the inhibition of protein synthesis, by Pm. It is proposed that aging human diploid fibroblasts contain altered ribosomes which react differently with Pm.

The accuracy of protein synthesis in reticulocyte and HeLa cell lysates

The accuracy of translation in protein synthesis is measured as the rate of misincorporation of a particular amino acid, different from that specified by an mRNA codon, into protein. The cowpea variant of tobacco mosaic virus, CcTMV, contains no cysteine or methionine in its coat protein. Translation in vitro of purified CcTMV coat protein mRNA by rabbit reticulocyte and HeLa cell lysates has been performed. The coat protein product was purified by immunoprecipitation with specific antisera, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The error rate was measured by comparing the incorporation of [35S]cysteine with incorporation of [3H]leucine, and the total CcTMV coat protein synthesized was calculated from its known leucine content. An error rate of (1-2) X 10(-3) cysteines/CcTMV coat protein was obtained with reticulocyte lysates. If errors were cysteine incorporation in place of arginine, this number is converted to 3 X 10(-4) cysteine/codon. If cysteine was incorporated anywhere in the polypeptide, the rate is 9 X 10(-6) cysteines/amino acid. The error frequencies with HeLa cell lysates were 6-fold higher. Paromomycin, a eukaryotic misreading antibiotic, increased error rates 10-fold in both lysates. These data compare well with in vivo measurements and suggest that some transformed cells may survive with higher mistranslation rates.

Clonal variations in keratin: Intermediate filament expression by human somatic cell hybrids

The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes.

Chapter 4 The Experimental Manipulation of Keratin Expression and Organization in Epithelial Cells and Somatic Cell Hybrids

Publisher Summary Keratins are found in nearly all epithelial tissue types in vertebrates, and in many cases keratins constitute the majority of the proteins produced ( for e.g., skin and its derivatives). Their organization in the cytoplasm of epithelial cells has made them the fundamental elements of the cytoskeleton and established them as a part of a much larger, interrelated group of polymer-forming proteins that give rise to intermediate filaments. Experimental manipulation of epithelial cells provides a means by which these proteins and the filaments formed by them can be better understood, both in the context of basic cell biology and with regard to their importance in the clinical diagnosis of disease. This chapter discusses some of the consequences of the cytological and genetic manipulation of keratin intermediate filaments in epithelial cells and somatic cell hybrids. The approaches presented are used to investigate keratin intermediate filaments with respect to the overall structure and function of the vertebrate cytoskeleton and to relate the expression of keratin genes and the subsequent organization of keratins to specific cell behavior and growth potential. Results and interpretations from two experimental approaches are presented in the chapter in which the expression and/or organization of keratins has been altered. The first involves the analysis of drug-induced alterations of cytokeratin in cultured epithelial cells and the second focuses on the analysis of clonal variations in keratin filament expression by human somatic cell hybrids.

Intermediate filament expression and lifespan potential in human somatic cell hybrids

SummaryLimited lifespan human diploid fibroblast cells have been fused with the HeLa derived cell line HEB 7A which possesses transformed growth characteristics and unlimited division potential. HEB 7A expresses keratin intermediate filaments, while the fibroblast cells express only vimentin intermediate filaments. Independently arising clones of hybrids were examined for the presence of keratin by indirect immunofluorescence. Of 11 limited lifespan hybrids, all were keratin negative and possessed the growth characteristics of the fibroblast parent. Of 8 transformed hybrids, 6 arising early after fusion and 2 arising late, all were keratin-positive and simultaneously expressed the transformed growth characteristics of loss of density dependent growth inhibition, low serum dependence, and anchorage independence. It is concluded that the growth properties of these hybrids are associated with the type of intermediate filament expressed. The intermediate filament expression is therefore a marker of proliferative potential in these hybrids.

Inherent resistance of HeLa cell derivatives to paromomycin

SummaryThe human tumor-derived cell line HeLa S3 and nuclear and mitochondrial gene mutants derived from it are resistant to the aminoglycoside antibiotic, paromomycin (PAR). Other carcinoma-derived cells, SV40-transformed cells, and four human diploid fibroblast cell lines are all sensitive to PAR. Sensitivity is dependent on cell density, and at cell numbers greater than 400/cm2 sensitive cells will proliferate in PAR. The resistance to PAR is inherited in a dominant manner in cell-to-cell fusion hybrids, but is not transferred in cytoplast-to-cell fusions. PAR resistance is therefore encoded by a nuclear gene(s). Resistance to PAR is not caused by changes in the response to mitochondrial or cytoplasmic protein synthesis to PAR in vitro. The uptake of PAR is similar in resistant and sensitive cells, and dimethyl sulfoxide does not render resistant cells more sensitive. Thus, HeLa cell PAR resistance is unlike previously reported ribosomal mutations and may derive from differences in the intracellular metabolism of PAR.