Darrell Salk

Werner's syndrome: A review of recent research with an analysis of connective tissue metabolism, growth control of cultured cells, and chromosomal aberrations

SummaryWerners syndrome is a rare, autosomal recessive condition with multiple progeroid features, but it is an imitation of aging rather than accelerated or premature senescence. Somatic chromosome aberrations occur in multipe tissues in vivo and in vitro, and there is an increased incidence of neoplasia. Thus, Werners syndrome can be classified in the group of chromosome instability syndromes. Recent findings provide additional support for the concept that there is aberration of connective tissue metabolism in Werners syndrome, but it is unclear whether this is a primary or secondary manifestation of the underlying genetic defect. Abnormal growth characteristics are observed in cultured skin fibroblast-like cells and this provides another avenue for current research. Identification of the basic genetic defect in Werners syndrome might clarify our understanding of the normal aging process in general, or might elucidate specific aspects such as the development of neoplasia, atherosclerosis, diabetes, or osteoporosis.

Cytogenetics of Werner’s syndrome cultured skin fibroblasts: variegated translocation mosaicism

Skin fibroblast-like (FL) cells from patients with Werners syndrome (adult progeria) regularly demonstrate frequent pseudodiploidy involving variable structural rearrangements that are clonal: variegated translocation mosaicism (VTM). Ninety-two percent of 1,538 metaphases from 29 independent strains derived from five patients with Werners syndrome demonstrated this cytogenetic abnormality. In contrast, only eight (8.4%) of 95 non-Werners syndrome FL cell cultures demonstrated VTM: seven with low-grade VTM (approximately 5% of 300 metaphases), and one with VTM affecting 90-100% of metaphases. Unlike the cytogenetic abnormalities observed in the terminal stages of normal FL cell cultures, VTM occurs throughout the entire lifespan of Werners syndrome cultures. Ten of the identifiable break points in 1,005 banded metaphases accounted for 27% of all definable rearrangements. Baseline sister chromatid exchanges were not increased. Cocultivation of Werners syndrome and normal strains did not induce VTM in the normal strain. The relationship between VTM and the reduced growth potential of Werners syndrome FL cells is not yet understood, nor is the relationship between these in vitro abnormalities and the presumptive single gene defect that causes the progeroid clinical manifestations of Werners syndrome.

Chapter 2. Morphological and Biochemical Heterogeneity of Amniotic Fluid Cells in Culture

Publisher Summary This chapter describes the morphological and biochemical heterogeneity of amniotic fluid cells in culture. Midgestation amniotic fluid cells constitute a precious source of human cells for the purpose of cell biological investigation. Several properties contribute to the diagnostic and scientific value of these cells, the most important of which are the presence in a single amniotic fluid specimen of multiple, morphologically and biochemically distinct cell types that are all derived from the fetus or from fetal membranes and thus are isogenic. It is found that in addition to variable amounts of cellular debris and anucleate particles, second-trimester amniotic fluid contains between 103 and 105 nucleated cells per milliliter of fluid. All epithelial surfaces in direct or indirect contact with the amniotic cavity are possible sites of origin of these cells. According to their behavior in the culture environment, nucleated amniotic fluid cells can be operationally divided into three separate categories. The first category includes cells that remain afloat even after prolonged periods of incubation. It is found that amniotic fluid F-type cells have the greatest in vitro growth potential of all colony-forming cells.

Systematic growth studies, cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblasts

SummaryThe growth of 20 independently derived skin fibroblastlike (FL) cell strains from three individuals with Werner syndrome (adult progeria) was compared with the growth of ten FL cell strains from normal individuals. Population growth rates and total replicative life spans of Werner syndrome strains averaged 55% and 27%, respectively, of the growth rates and life spans of non-Werner strains. In the first few passages, four Werner syndrome strains demonstrated population growth rates in the low normal range, but the longest-lived Werner syndrome strain had only 75% of the total replicative potential of the shortest-lived normal strain. Exponential growth rates, cloning efficiencies, and saturation densities of Werner strains were also reduced, whereas cell attachment was normal. Viable cells (identified by dye exclusion) were maintained in post-replicative Werner syndrome and control cultures for periods of at least 10 months; there was no evidence of accelerated post-replicative senescence or cell death of Werner syndrome FL cells. Cocultivation of Werner syndrome and normal strains did not influence population growth rates of either strain. Two proliferating hybrid clones were obtained from fusions of normal and Werner syndrome FL cell strains and these hybrids displayed the reduced growth potential typical of Werner syndrome FL cells. These studies confirm that low growth rate and sharply reduced replicative life span are characteristic of cultured skin FL cells from patients with Werner syndrome, and they suggest that these characteristics are not affected by complementation with non-Werner FL cells.

Growth Characteristics of Werner Syndrome Cells in Vitro

Cultured skin fibroblast-like (FL) cells from patients with Werner syndrome grow poorly: reduced growth potential has been reported by more than 11 laboratories for cultures from more than 26 patients, using many different tissue culture media. This characteristic is of importance to research in Werner syndrome for several reasons. It remains to be determined whether poor growth is a primary or a secondary manifestation of the basic molecular defect, and the relationship is not yet clear between reduced growth of cultured skin fibroblasts and the other manifestations of Werner syndrome in vitro and in vivo. This characteristic also makes it difficult to obtain sufficient growth for clonal studies or for biochemical studies that require a large number of cells.

Evidence of clonal attenuation, clonal succession, and clonal expansion in mass cultures of aging Werner’s syndrome skin fibroblasts

Skin fibroblast-like (FL) cells from patients with Werners syndrome (adult progeria) demonstrate multiple, stable, structural chromosome rearrangements (variegated translocation mosaicism) which can be used to identify cytogenetically marked clones of cells within a mass culture. We have cytogenetically followed eight FL cell strains (from two patients) throughout their entire in vitro replicative lifespans, and we show a correlation between the expansion and attenuation of individual clones and the growth of the mass cultures. One strain, which was aged several times, demonstrated a generally reproducible pattern of clonal succession but, surprisingly, also demonstrated, in two parallel derivative cultures, the late emergence of two relatively rapidly growing clones that had not been observed in the parental culture. These observations suggest that clonal succession and clonal attenuation occur in mass cultures, as had been predicted on the basis of dilute-plating cloning experiments. Our results may have implications for models of in vitro cellular senescence. In addition, there are interesting parallels with the tissue hyperplasia associated with in vivo aging, and this observation is compatible with the suggestion that skin FL cells in vitro provide a model for hyperplasia in vivo.

Separation of retinoblastoma and esterase D loci in a patient with sporadic retinoblastoma and del(13)(q14.1q22.3)

SummaryA chromosome 13 deletion in a patient with sporadic retinoblastoma appears to have separated the loci for retinoblastoma and esterase D. This study indicates that: (1) the retinoblastoma locus is distinct from the esterase D locus; and (2) the linear order of these genes is centromere-esterase D-retinoblastoma.

Cytogenetic Aspects of Werner Syndrome

Cultured skin fibroblast-like (FL) cells from patients with Werner syndrome display frequent pseudodiploidy involving multiple, variable structural chromosome rearrangements that are clonal (Salk et al., 1981a). This cytogenetic abnormality, which has been called variegated translocation mosaicism (VTM), has also recently been observed in peripheral blood lymphocytes (Scappaticci et al., 1982). Werner syndrome may properly be classified as a chromosome instability syndrome because, like Bloom syndrome, ataxia telangiectasia, Fanconi anemia, and xeroderma pigmentosum, it is autosomal recessive, displays chromosome instability, and is associated with an increased incidence of neoplasia.

Werner's syndrome and human aging

Historical Perspective.- On Cataract in Conjunction with Scleroderma.- Werners Syndrome (Progeria of the Adult) and Rothmunds Syndrome: Two Types of Closely Related Heredofamilial Atrophic Dermatoses with Juvenile Cataracts and Endocrine Features a Critical Study with Five New Cases.- Werners Syndrome: A Review of its Symptomatology, Natural History, Pathologic Features, Genetics and Relationship to the Natural Aging Process.- Werner Syndrome: A Review of Recent Research with an Analysis of Connective Tissue Metabolism, Growth Control of Cultured Cells, and Chromosomal Aberrations.- Clinical, Pathological and Genetic Aspects of the Werner Syndrome and Normal Human Aging.- Genetics and Aging: Werners Syndrome as a Segmental Progeroid Syndrome.- Clinical, Endocrine and Metabolic Aspects of the Werner Syndrome Compared with Those of Normal Aging.- Pathology of the Werner Syndrome.- Neuropathology of the Werner Syndrome.- Werners Syndrome and Aging: A Reappraisal.- A Comparison of Adult and Childhood Progerias: Werner Syndrome and Hutchinson-Gilford Progeria Syndrome.- Clinical, Demographic and Genetic Aspects of the Werner Syndrome in Japan.- Immunological Aspects of the Werners Syndrome: An Analysis of 17 Patients.- Clinical and Metabolic Studies on the Werners Syndrome: With Special Reference to Disorders of Lipid and Liver Function.- Growth Control of Werner Syndrome Cells In Vitro.- Growth Characteristics of Werner Syndrome Cells in Vitro.- Studies of SV40-Infected Werner Syndrome Fibroblasts.- Experimental Studies on Werners Syndrome Fibroblasts.- Cell Fusion Studies in the Werner Syndrome.- Cell Fusion Studies and Biochemical Analysis of DNA Synthesis in Werner and Non-Werner Cultured Cells.- Molecular Mechanisms of Cellular Senescence: DNA Metabolism and Chromatin Change.- Histone H1 in G1 Arrested, Senescent and Werner Syndrome Fibroblasts.- Genome Reorganization During Aging of Dividing Cells.- Cellular Mechanisms of Ageing in the Werner Syndrome.- Autoradiographic Studies of DNA Replication in Werners Syndrome Cells.- Abnormal Fibroblast Aging and DNA Replication in the Werner Syndrome.- Extrachromosomal Circular DNA and Aging Cells.- Molecular Mechanisms of Cellular Senescence: Errors in Protein Synthesis.- Protein Synthetic Fidelity in Aging Human Fibroblasts.- Analysis of Cellular Senescence through Detection and Assessment of RNAs and Proteins Important to Gene Expression: Transfer RNAs and Autoimmune Antigens.- Cytogenetics of the Werner Syndrome.- Cytogenetic Aspects of Werner Syndrome.- A Population and Cytogenetic Study of the Werner Syndrome in Sardinia.- Connective Tissue Metabolism and Disturbances in the Werner Syndrome.- Proteoglycans in the Werner Syndrome and Aging: A Review and Perspective.- Cell Surface Changes in Senescent and Werners Syndrome Fibroblasts: Their Role in Cell Proliferation.- Acidic Glycosaminoglycans in Werners Syndrome: Studies on Levels in Tissue, Organ, Cell and Fluid.- Acidic Glycosaminoglycans of SV40-Transformed Werners Syndrome Cells.- Glycosaminoglycan Synthesis in Untransformed and Transformed Werner Syndrome Fibroblasts: A Preliminary Report.- Gene Action, Development and Aging: New Directions for Research in the Werner Syndrome.- The Notion of Primordial Building Blocks in Construction of Genes and Transcriptional and Processing Errors due to Random Occurrence of Oligonucleotide Signal Sequences.- Appearance of Albumin-Producing Cells in the Liver of Anabulminemic Rats on Aging and Administration of Mutagens.

INDUCTION OF LONG-TERM IMMUNITY TO PARALYTIC POLIOMYELITIS BY USE OF NON-INFECTIOUS VACCINE

Naturally acquired immunity to paralytic poliomyelitis, which is of long duration, is associated with serum antibody, immunological memory, or both. Persistence of circulating antibody is reassuring but not essential for long-term protection. Immunological memory induced by killed poliovirus vaccine is similar to that induced by infection, which is not a prerequisite for the induction of long-term immunity. Animal studies of experimental vaccines indicate that some antigenic components of poliovirus induce immunological memory without producing detectable antibody; killed poliovirus vaccine has the same effect in man. Killed vaccine containing 40, 8, and 32 D-antigen units of poliovirus types 1, 2, and 3, respectively, protects all recipients in both one-dose and two-dose schedules. This means that either schedule can be chosen to fit in with local immunisation programmes and thus reduce costs and increase population coverage.