Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

Early and late replication of chromosomes in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes were analysed using pulse and continuous incorporation of ³H-thymidine. In both types of cells chromosomes begin the synthesis period synchronously. The termination of the DNA synthesis of chromosomes is an asynchronous process and has peculiarities in every type of cells, chromosomes 1, 16, 21–22 are labelled relatively more intensively in fibroblast cultures than in leucocytes but the chromosomes of group 4–5, on the contrary, contain more grains in leucocyte cultures than in fibroblast cultures. It is highly probable that the discovered differences in late replication of the mentioned chromosomes in the investigated types of cells are connected with the differences in functioning of these chromosomes in the two different types of cell systems.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

By autoradiography with ³H-thymidine and ³H-deoxycytidine it is shown that chromosomes 1 and 16 in cultures of embryonic fibroblasts at the termination of the S period synthesise AT- and GC-rich DNA at different rats: in both chromosomes the labelling of AT-bases is more intensive. In leucocyte cultures both nucleotide pairs label equally in these chromosomes. Chromosomes 2, 3, 4–5 and 21–22 are labelled equally in both cultures with respect to AT-and GC-pairs. Fibroblasts and leucocytes differ in the relative intensity of DNA synthesis at the end of the S period: chromosomes 1,16 and 21–22 contain more label in the case of fibroblasts (chromosome 1 solely due to AT-pairs) and chromosome 4–5 in the case of leucocytes. Analysis of distribution of late label along chromosome 1 showed that in fibroblast cultures the pericentromeric regions of both arms are labelled more intensively in respect to both nucleotide pairs than in leucocyte cultures. Both in fibroblast and leucocyte cultures no significant distinctions in the distribution of AT-and GC-pairs along chromosome 2 were established. In fibroblast cultures the pericentromeric regions of both arms of chromosome 3 are labelled more intensively than other regions. In leucocyte cultures the pericentromeric region of the short arm of this chromosome is labelled with the same intensively as in fibroblasts, whereas in the pericentromeric region of the long arm the intensity of incorporation of labelled synthesis precursors decreases. — Analysis of results obtained in the present study together with data of previous studied (Slesinger et al., 1974; Lozovskaya et al., 1976; Lozovskaya et al., 1977) shows that differences between the two types of cells in the intensity of late ³H-thymidine labelling in the C-heterochromatin regions of chromosomes 1 and 16 may be explained both by variation of replication time in leucocytes as compared with fibroblasts and by variation of the content of AT- rich DNA. Differences observed in other chromosomes are probably due to different times of replication of these chromosomes in leucocytes and fibroblasts. — Thus, the process of cell system differentiation involves not only differential activity of the genome (the main mechanism) that is connected with differences in the replication time of chromosomes and of their regions but also variation of the quantity of genetic material.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

Distributions of AT- and GC-base pairs along the length of chromosomes 1, 2, 3 and 16 in primary cultures of embryonic fibroblasts and of peripheral blood leucocytes were studied by autoradiography with: 1. ³H-thymidine and ³H-deoxycytidine; 2. ³H-deoxyadenosine and ³H-deoxyguanosine. It has been shown that the two types of cells differ in the DNA content and proportion of AT- and GC-nucleotide pairs in the centromeric heterochromatin of chromosome I: this region contains more DNA in fibroblasts than in leucocytes mainly due to AT-pairs. In both types of cells the telomeric region of the short arm of this chromosome contains more GC- than AT-pairs. Similar results were obtained for C-heterochromatin of chromosome 16: the frequencies of labelling of this region by ³H-deoxyadenosine and ³H-thymidine in fibroblast cultures were higher than in case of ³H-deoxycytidine and ³H-deoxygyanosine, and in leucocyte cultures these frequencies were almost equal. No differences in the distributions of base pairs along the length of chromosome 2 and 3 were established in the two types of cells. — The nature of the established phenomenon may be connected with under-replication or loss in another way of part of the genetic material in the process of development and differentiation of cell systems.     

DNA replication sequence of human chromosomes in blood cultures

Summary The pattern of labelling over the chromosomes, the chronology of chromosome duplication and the duration of the S and G ₂ periods in the leukocytes from 6 normal females and 5 normal males, have been studied by using a combination of pulse and continuous tregtments with thymidine-H³. According to the criteria used to analyse the results it is suggested that the S period begins 15 to 20 hours and finishes 5 to 3 hours before the cells reach the metaphase stage. The S period could be subdivided into the four phases S₁ to S₄.The first chromosomes to replicate were Nos. 1, 3, 5 and X followed by the Nos. 2, 4 and several chromosomes of groups 6–12, 13–15 and 19–20. Later the pairs 16, 17, 18 and the chromosomes of group 21–22 replicated. Chromosome Y in the male was the last to replicate, beginning its duplication when all the other chromosomes had reached the intermediate S stage.The earliest chromosomes to finish the duplication were Nos. 19, 20 and 21 followed by Nos. 16, 17, 18, 22 and the chromosomes of group 13–15. Afterward and at about the same time the replication of pairs 2, 4, 6, 8, the X and Y chromosomes in the male and one X chromosome in the female concluded. The other X chromosome in the female was the last to end its duplication appearing totally labelled until the final stage of the S period.Replication of the long and medium size chromosomes begins at localised regions, then extends over the total length of the chromosome and at the end of the S stage takes place only in small zones different from those replicating early.Asynchrony between homologous chromosomes was observed at the beginning and at the end of the S period.     

Asynchrony in late replication between homologous autosomes in primary cultures of Chinese hamster fibroblasts

Asynchronies in late replication of the autosomal chromosome pair No. 5, and to some extent of pair No. 4, were found after thymidine pulse labeling cultures of partially synchronized Chinese hamster lung fibroblasts from nine to nine and a half hours and from nine and a half to ten hours after block removal. In contrast to this, no asynchrony could be detected in the replication of homologous autosomes after continuous labeling for the last two hours of the S-phase. — G-banding and C-banding revealed no differences between the homologous autosomes. — These findings indicate that besides the known form of asynchronous replication in mammalian cells during S-phase on the chromosomal level, there also exists an asynchronous replication between homologous autosomes of the same complement.     

Human embryonic fibroblasts support single cell enzymatic expansion of human embryonic stem cells in xeno-free cultures

The future application of human embryonic stem cells (hESC) for therapeutic approaches requires the development of xeno-free culture conditions to prevent the potential transmission of animal pathogens or xenobiotic substances to hESC. An important component of the majority of hESC culture systems developed is the requirement for fibroblasts to serve as feeders. For this purpose, several studies have used human foreskin fibroblasts established under xeno-free conditions. In this study we report xeno-free establishment and maintenance of human embryonic fibroblasts (XHEF) and demonstrate their ability to support long-term self-renewal of hESC under xeno-free culture conditions, using a commercially available complete medium. Importantly, our culture conditions allow enzymatic passaging of hESC. In contrast, hESC cultured on human foreskin fibroblasts (XHFF) under the same conditions were poorly maintained and rapidly subject to differentiation. Our study clearly shows that the source of human fibroblasts is essential for long-term xeno-free hESC maintenance.     

Clastogenic effects of zinc chloride on human peripheral blood leucocytes in vitro

The clastogenic effects of three different concentrations of zinc chloride on human peripheral blood leucocytes were studied in vitro. The highest concentration (1.5 x 10(-3) M) was lethal after 48 and 72 h of culture and no blast cells were formed. The two lower concentrations (3.0 x 10(-4) M and 3.0 x 10(-5) M) significantly reduced the frequency of cell division, induced chromatid breaks and damaged cells in frequencies significantly higher than in control experiments maintained in sodium chloride and in distilled water.     

Antigen-induced and polyclonal B-cell responses in human peripheral blood lymphocyte cultures

This work was designed to delineate the anti-hapten antibody (Ab) response induced by trinitrophenol-polyacrylamide (TNP-PAA) beads from the nonspecific B-cell response which concomitantly occurs in human peripheral blood mononuclear cell (PBMC) cultures. Indeed human PBMC produce consistent amounts of immunoglobulins when cultured at high cell density in the presence of fetal bovine serum, regardless of the presence of antigen. In contrast, the stimulation of such cultures by TNP-PAA leads to an Ab response characterized by the following: cells secreting anti-hapten Ab at a high rate (detected by a plaque-forming cel (PFC) assay); a 10-30 times enhancement in the number of hapten-specific binding cells (detected by a rosette-forming cell (RFC) assay); the production of anti-TNP IgM Ab (detected by an ELISA assay). The anti-TNP response is specifically triggered by the particulate antigen, as shown by the following: The TNP-PAA antigen induces a clear-cut increase in the amount of anti-TNP Ab whereas it only marginally increases that of total IgM. The anti-TNP Ab response is specifically abolished when anti-TNP RFC are depleted from the PBMC preparation before the initiation of the cultures. The anti-TNP Ab response is specifically abolished when PBMC are triggered by TNP-PAA in the concomitant presence of a soluble TNP-protein conjugate. These results demonstrate the ability of polymeric antigens to specifically activate human peripheral blood B cells.     

Actin content and organization of microfilaments in primary cultures of mouse embryonic fibroblasts (in vitro ageing)

Actin distribution in serially passaged embryonic mouse fibroblasts has been visualized by the anti-actin-PAP method; the organization of the microfilaments has been observed by electron microscopy (SEM and TEM). Four successive actin patterns have been identified: early (few well-organized bundles of microfilaments), middle-aged (many well-organized bundles and patches around the nucleus), late (numerous ill-organized filamentous structures and diffuse perinuclear-actin) and "senescent" (heavy packs of short microfilaments around the nucleus). All the observed actin-positive filaments were disrupted by cytochalasin B treatment. The cytoplasmic actin complex was cell-age and not cell-size-dependent; it behaved differently from the cytoplasmic microtubular complex to serially subcultivated fibroblasts. Measurements of the cell-protein content (Lowry's method) and SDS-polyacrylamide gel electrophoresis (Laemmli's method) have been performed in the successive population doubling levels (PDL) of the primary cultures. Triton-insoluble actin increased in parallel with total protein and reached about 4% of the total proteins in all the PDLs. Triton-soluble actin also increase at the beginning of the middle-aged period (generally 6 PDL) and another in declining cultures (generally 10 PDL). Total actin amounted to about 8% of the total proteins in early fibroblasts, to about 16% at the beginning of the middle-aged period and to about 20% in the declining terminal cultures. Taking into account all the known characteristics of subcultivated primary cultures, we tentatively consider the evolution of the fibroblasts as an in vitro differentiation followed by true in vitro senescence in the declining cultures. Regarding the cytoplasmic actin-complex, senescence would be characterized by a sharp increase in soluble actin, an unbalanced ratio between soluble and insoluble actin and an impairment of the ability of the microfilaments to form well-organized bundles.     

Comparative study of different types of rosette-forming cells in human peripheral blood and tonsils]

E-, EA-, and EAC-reaction of rosette formation and detection of surface immunoglobulins were applied to the comparative study of the peripheral blood lymphocytes populations and tonsils in man. The relative lymphocyte content with different surface characteristics differed significantly in these populations. In detection of surface immunoglobulins on EAC-rosette forming cells it was revealed that not all the C3-lymphocytes of the tonsils bore surface Ig. A possibility of appearance of the C3-receptor on the activated T-lymphocytes is discussed.     

Domain structure of proteoheparan sulphate from confluent cultures of human embryonic skin fibroblasts.

Radiolabelled proteoheparan sulphates were isolated from confluent monolayers of fibroblasts and from their spent media. The cell-surface-associated proteoglycan (Mr 350 000) has a core protein of Mr 180 000 that is cleaved by reduction of disulphide bonds into polypeptides of Mr 90 000, both of which can bind transferrin [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661]. Thrombin digestion of the proteoglycan yielded two major fragments. The larger one contained the heparan sulphate chains and glycoprotein-type oligosaccharides, whereas the smaller one contained interchain disulphide bond(s) and had affinity for transferrin as well as for octyl-Sepharose. The larger thrombic fragment was cleaved by trypsin into fragments containing the heparan sulphate chains and the oligosaccharides respectively. The smaller proteoheparan sulphate derived from the culture medium (Mr 150 000) had a core protein of Mr 30 000, which contained heparan sulphate-attachment and oligosaccharide-attachment regions, but no domains for binding of transferrin or for hydrophobic interactions.     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.