Synthesis of sulfated proteoglycans throughout the cell cycle in smooth muscle cells from pig aorta

Cultured smooth muscle cells from pig aorta arrested in G0 phase by serum deprivation were stimulated to proliferate by replacing the medium with one containing 10% serum. Studies in DNA replication and proliferation of cells showed a relatively good synchrony: 90% of the cells were in G1 phase for 16 h after addition of serum; they entered S phase between 18 and 24 h, completed S phase and traversed G2 phase between 24 and 30–32 h; 75% of these cells multiplied after 30–32 h and the remainder were blocked at the end of G2 phase. The synthesis and secretion of sulfated proteoglycans were examined throughout a full cell cycle using metabolic labelling with [³⁵S]sulfate. Smooth muscle cells in G1 or G2 phase synthesized and secreted sulfated proteoglycans with a possible pause at the end of the G2 phase but at the beginning of the S phase and during mitosis the incorporation of [³⁵S]sulfate into these macromolecules stopped entirely. Structural characteristics of sulfated proteoglycans secreted into the medium during G1 phase and an entire cell cycle were investigated. The proportion of proteoglycan complexes and the relative hydrodynamic size of monomers and of constituent subunits of complexes were determined after chromatography on Sepharose CL-2B and CL-6B columns run under both associative and dissociative conditions. No significant differences were observed for the periods of the cell cycle that were studied:

1. 1. [³⁵S]Proteoglycan complexes represented at the end of G1 phase and of the cell cycle respectively 19 and 16% of the total [³⁵S]proteoglycans secreted into the medium.

2. 2. More than 90% of the subunits, obtained after dissociation of complexes, were characterized by a similar k_av after chromatography on Sepharose CL-2B columns eluted under dissociative conditions (k_av 0.68 at the end of G1 phase and 0.65 at the end of full cell cycle).

3. 3. About 95% of monomers synthesized at the two stages of the cell cycle were eluted at k_av 0.25 after chromatography on Sepharose CL-6B column run under associative conditions and were characterized by a similar glycosaminoglycan distribution. These results suggest that smooth muscle cells in culture liberate similar populations of proteoglycans into the medium during the G1 and G2 phases.

     

Identification of external membrane proteins of the T lymphoblast cell line CCRF-CEM

The 70 membrane proteins of the T lymphoblast cell line CCRF-CEM were characterized by

1. 1. [³⁵S]methionine internal radiolabeling;

2. 2. [¹²⁵I]iodine labeling by a lactoperoxidase-mediated method;

3. 3. [³H]fucose internal labeling;

4. 4. binding to a lentil lectin adsorbant column;

5. 5. susceptibility to digestion with limited amounts of papain.

Of the three methods of radiolabeling membrane proteins, [³⁵S]methionine best displayed all proteins although some individual proteins were heavily iodinated or fucosylated. Thirty proteins were externally exposed as defined by susceptibility to lactoperoxidase-mediated radio-iodination and to digestion with minute amounts of papain. Thirtyfive proteins were bound to a lentil lectin absorbant column. p44 (HLA-A and -B antigens) were iodinated, fucosylated, susceptible to papain digestion and bound to the lectin column. β₂-Microglobulin was iodinated and bound to the lectin column. The identifications and functions of other membrane proteins were not known. In general, proteins of high molecular weight (100 000 to 250 000 D) were more heavily radio-iodinated and fucosylated than were proteins of lower molecular weights. p95 was the most heavily fucosylated protein, p110, which had been identified only on T lymphoblasts, was fucosylated and was iodinated. p65, which was found only on the T lymphoblast line CCRF-CEM and could represent a lymphocyte subpopulation-specific molecule, was iodinated and fucosylated. p15 and p18 were equally and densely labeled with [³⁵S]methionine but only p18 was fucosylated and it was heavily radio-iodinated. These experiments help to define the external membrane proteins of a T lymphoblast cell line in part for the selection of proteins for isolation in order to raise antisera for immunodiagnostic and functional studies.     

Cytotoxic effects of dexamethasone restricted to noncycling,early G1-phase cells of L1210 leukemia

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10^?6-10^?5 M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to ³H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.     

Asymmetry in the renewal of molecular classes of phosphatidylcholine in the rat-erythrocyte membrane

1. 1. Rat-blood phospholipids were labeled in vivo with [³²P]phosphate. The erythrocytes were treated with phospholipase A₂ plus sphingomyelinase to discriminate between the labeling patterns of the phospholipids from the inner and outer layer of the membrane.

2. 2. The specific activities of the more unsaturated classes of phosphatidylcholine were higher in the outer layer of the erythrocyte membrane than in the inner layer. The disaturated class, however, had the highest specific activity in the inner layer.

3. 3. After incubating ³²P-labeled erythrocytes in unlabeled plasma, the labeling pattern recovered in the molecular classes of plasma phosphatidylcholine was very similar to that of the phosphatidylcholines in the outer layer of the erythrocyte membrane.

4. 4. It is proposed that the exchange of phosphatidylcholines between plasma and the outer layer of the erythrocyte is mainly responsible for the renewal of the unsaturated phosphatidylcholines of the erythrocyte, and that the acylation activity of the erythrocyte is directed towards the formation of disaturated phosphatidylcholines at the inside of the membrane.

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

Successive 3H and 14C labeling of DNA in a stimulus-response system

Quiescent (G0) cells of the central zone region of the rat lens epithelium were recruited into the cell cycle by a wound stimulus. Cells were pulsed with labeled DNA precursor at several different times after the initiation of the DNA synthesis response to wounding and allowed to progress into the mitotic phase. Analysis of mitotic figures resulted in PLM (percentage labeled mitoses) curves that indicated a G2 duration of about 6 h. Double isotopic labeling ([3H]thymidine followed by [14C]thymidine) was utilized to demonstrate the completion of DNA synthesis in earliest responders. Cells completed DNA synthesis in less time (3-5 h) than reflected by the approximately 8-h widths of PLM curves. This discrepancy is attributed to the uptake and retention of labeled precursor by the stimulus-responsive cells while they are still in a pre-S phase condition. Based on a comparison of transit times through G2 and of labeling times to midpoint appearances of labeled mitotic figures, earlier responders do not appear to have faster rates of cell cycle progression than cells responding 2-4 h later. G2 transit time is also comparable for central zone lens cells responding to the relatively strong stimulus of wounding and for the nonperturbed cells previously studied in the germinative zone of the lens epithelium.     

Synchronization of mouse 3T3 and SV40 3T3 cells by way of centrifugal elutriation

Populations of G1 phase 3T3 and SV40 3T3 mouse fibroblasts have been isolated from exponentially growing cultures by the technique of centrifugal elutriation. Return of the G1 phase cells to growth conditions results in their synchronous passage through the cell cycle, as determined from monitoring of cell number, [³H]thymidine ([³H]TdR) incorporation and fraction of [³H]TdR labeled nuclei. The durations of G1, S and G2 phases are consistent with values obtained by previous investigators using conventional induction techniques for synchronization. The method for isolation of the G1 phase cells is rapid, the yield is high and the process does not appear to alter the temporal aspects of the cell cycle in either cell type.     

Efficient In Vitro Labeling Rabbit Bone Marrow-Derived Mesenchymal Stem Cells with SPIO and Differentiating into Neural-Like Cells

Mesenchymal stem cells (MSCs) can differentiate into neural cells to treat nervous system diseases. Magnetic resonance is an ideal means for cell tracking through labeling cells with superparamagnetic iron oxide (SPIO). However, no studies have described the neural differentiation ability of SPIO-labeled MSCs, which is the foundation for cell therapy and cell tracking in vivo. Our results showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) labeled in vitro with SPIO can be induced into neural-like cells without affecting the viability and labeling efficiency. The cellular uptake of SPIO was maintained after labeled BM-MSCs differentiated into neural-like cells, which were the basis for transplanted cells that can be dynamically and non-invasively tracked in vivo by MRI. Moreover, the SPIO-labeled induced neural-like cells showed neural cell morphology and expressed related markers such as NSE, MAP-2. Furthermore, whole-cell patch clamp recording demonstrated that these neural-like cells exhibited electrophysiological properties of neurons. More importantly, there was no significant difference in the cellular viability and [Ca²⁺]i between the induced labeled and unlabeled neural-like cells. In this study, we show for the first time that SPIO-labeled MSCs retained their differentiation capacity and could differentiate into neural-like cells with high cell viability and a good cellular state in vitro.     

An 18 000-dalton protein metabolically labeled by polyamines in various mammalian cell lines

The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [¹⁴C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoreses. Under such incubation conditions, a single protein band with an apparent molecular weight of 18 000 was radioactively labeled. [¹⁴C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of γ-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [¹⁴C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation.     

Temporal and Quantitative Expression of the Myelin-Associated Lipids,Ethanolamine Plasmalogen,Galactocerebroside, and Sulfatide,in the Differentiating CG-4 Glial Cell Line

We determined the expression of three myelin-typical lipids in the continuous CG-4 glial cell line of oligodendrocyte progenitor cells, as the cells differentiated into oligodendrocytes. On 6 different days during the first 9 days of oligodendrocyte development, cells were labeled for 24 h with [³H]ethanolamine to label ethanolamine plasmalogens or with [³H]galactose to label the galactocerebroside and sulfogalactocerebroside; and the amount of labeled lipid expressed on each day was determined. Each labeled lipid was expressed with its own specific time course and in a defined amount on each day of differentiation. Increased labeling of plasmalogens and sulfogalactocerebroside started at early developmental stages, and increased labeling of galactocerebroside started at later stages. The results indicate that the differentiating CG-4 cell line provides a valuable system to investigate factors affecting the early time course of myelin-lipid expression and the amounts expressed.     

Variation In G1 Transit Time Relative to the Cycloheximide and Actinomycin D Drug Restriction Points

The transit time distribution at various points in the cell cycle of synchronized Chinese hamster ovary cells was determined from the mitotic index, [³H]thymidine labeling index and increase in cell number monitored at regular intervals after mitotic selection. Variation in G₁ transit time compared with that for the total cell cycle indicates that variation in cell cycle transit time occurs mainly during G₁ phase. the cycloheximide (5.0 μg/ml) and actinomycin D (3.0 μg/ml) restriction points occur 0.2 and 1.7 hr prior to entry into S phase, respectively. the transit time distributions are further characterized by the moments of the distributions. the variance (2nd moment about the mean) of the transit time distribution at the actinomycin D restriction point is similar to the variance of the transit time distribution at the G₁/S border, thus variation in cell cycle transit time originates earlier than 1.7 hr prior to entry into S phase (i.e., the first 3/4 of G₁). If G₁ transit time variability and cell cycle control are related, then the results presented here indicate that the major regulatory events do not occur during late G₁ phase.     

CELL LOSS AND INFLUX OF LABELED HOST CELLS IN THREE TRANSPLANTABLE MOUSE TUMORS USING [125I]UdR RELEASE

The [¹²⁵I]UdR loss technique was used to estimate cell loss from RIF-1, EMT6 and KHJJ tumors in order to determine the length of the delay between labeling and the beginning of the loss of labeled cells, and also to calculate a value for ø, the cell loss factor. To determine the importance of reutilization of label released from the gut and/or the influx of labeled host cells, the blood flow to some tumors was occluded during and for 30 min after injection of the label. Relatively small amounts of radioactivity entered occluded RIF-1 tumors during 9 days after injection of [¹²⁵I]UdR, indicating that reutilization of systemic label and influx of labeled host cells are not significant in this system. In contrast, substantial amounts of radioactivity entered occluded EMT6 and KHJJ tumors, reaching 40% of the total activity in non-occluded tumors during 6 days following injection. After corrections were made for this influx of label, the [¹²⁵I]UdR loss curves from RIF-1 and EMT6 tumors were essentially exponential from the first day following injection of label. This was interpreted as indicating the loss of proliferating as well as non-proliferating cells from both tumors. The cell loss factor derived from the [¹²⁵I]UdR loss curves corrected for influx appeared to agree well with published values derived from analysis of percent labeled mitoses curves. In contrast, the corrected [¹²⁵I]UdR loss curves from KHJJ tumors showed that loss of activity began three days after injection of label, indicating that primarily nonproliferating cells are lost from this tumor.     

Complex Glutamate Labeling from [U-13C]glucose or [U-13C]lactate in Co-cultures of Cerebellar Neurons and Astrocytes

Glutamate metabolism was studied in co-cultures of mouse cerebellar neurons (predominantly glutamatergic) and astrocytes. One set of cultures was superfused (90 min) in the presence of either [U-¹³C]glucose (2.5 mM) and lactate (1 mM) or [U-¹³C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-¹³C]lactate (1 mM) and glucose (2.5 mM) for 4 h. Regardless of the experimental conditions cell extracts were analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy. ¹³C labeling of glutamate was much higher than that of glutamine under all experimental conditions indicating that acetyl-CoA from both lactate and glucose was preferentially metabolized in the neurons. Aspartate labeling was similar to that of glutamate, especially when [U-¹³C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-¹³C]lactate. The first part of the pyruvate recycling pathway, pyruvate formation, was detected in singlet and doublet labeling of alanine under all experimental conditions. However, full recycling, detectable in singlet labeling of glutamate in the C-4 position was only quantifiable in the superfused cells both from [U-¹³C]glucose and [U-¹³C]lactate. Lactate and alanine were mostly uniformly labeled and labeling of alanine was the same regardless of the labeled substrate present and higher than that of lactate when superfused in the presence of [U-¹³C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized. Special issue dedicated to John P. Blass.     

DEVELOPMENTAL ASPECTS OF ROOTS OF PISUM SATIVUM INFLUENCED BY THE G2 FACTOR FROM COTYLEDONS

A G2 Factor present in cotyledons of Pisum sativum influences several developmental events in roots. G2 Factor present in dry seeds (cotyledons and radicles) is transported to roots after germination and promotes cell arrest in G2 in about 35% of all root meristem cells. Present evidence suggests the G2 Factor promotes cell arrest in G2 only in cells that undergo normal cell differentiation (arrest) because the proportion of cells labeled with ³H-TdR after 16 hr does not differ among both seedlings or excised roots in the presence or absence of this substance. In this manner, trigonelline differs from chalones of animal tissues that usually suppress cell proliferation by cell arrest either in G1 or in G2. Experimental results suggest that cortex cells and not cells of vascular tissues in mature root tissues (20–22 mm from the meristem) are influenced by G2 Factor. Other recent publications indicate that the G2 Factor is trigonelline (N-methyl nicotinic acid) and concentrations of synthetic trigonelline from 10⁻⁵ to 10⁻⁷ m are effective in promoting cell arrest in G2 in one of the G2 Factor bioassays.     

Regulation of glycolipid synthesis during differentiation of clonal murine muscle cells

The two clonal murine muscle cell lines G7 and G8, originally derived from the M114 line [20], represent unique models for comparative studies of myogenesis. Glycolipid synthesis was examined during differentiation using [³H]-galactose and [³H]-glucosamine as precursors. Upon G7 contact glucosylceramide labeling increased and nLcOse₅Cer labeling stopped. During membrane fusion, glucosylceramide labeling stopped and lactosylceramide became the major synthetic product. G8 cells presented a different pattern, with increased labeling of GbOse₃Cer during myogenesis. The major ganglioside synthesized by both myoblasts was GM3, and more complex structures were observed following completion of myotube formation. Total glycopeptide labeling increased when G8 myoblasts fused and remained elevated in myotubes, whereas no differences during fusion of G7 cells were noted. Upon comparison of the two clonal lines, the only consistent observation was a significant increase in the synthesis of total gangliosides and neutral glycolipid during cell contact and membrane fusion (p < 0.02). The results suggest that changes in the synthesis of specific glycolipid structures during myogenesis are unique to each muscle cell line examined. However, transient increases in synthesis of total myoblast gangliosides and neutral glycolipids may be a more general phenomenon, possibly by curbing proliferation or by altering myoblast membrane fluidity characteristics during differentiation.Abbreviations MG6 VI³NeuAc-V⁴Gal-IV³GlcNAc-nLcOse₄Cer - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - Gal galactose - GlcNH glucosamine - PBS phosphate buffered saline - CK creatine kinase     

State-vector models of the cell cycle 1. Parametrization and fits to labeled mitoses data

In this paper we study the Hahn model of the cell cycle from the point of view that a cell population's age distribution is more relevant to labeled mitoses data than is the distribution of its transit times.Closed-form relationships are derived between the transition probabilities of the Hahn model and the transit time of the mean of a cohort of labeled cells (with the variance of their transit time through mitosis). Constraints result which define the acceptable values for the number of ages in the state vector and the length of the time step (rarely does the dimension of the state vector equal the number of time steps in the generation time).A generalization to distinct probabilities for G₁, S and G₂M is presented, and the automatic fitting of fraction-labeled mitoses (FLM) data is described. The doubling time of the population is used to define the daughter factor, via the largest eigenvalue of the state transition matrix. The performance of the generalized Hahn model is compared to that of other commonly used fitting methods using two sets of FLM data from the literature. The synthesis of continuous labeling curves is discussed as an independent check of the parametrization. Based on the stable age distribution resulting from fits to experimental FLM data, it is shown that a nonlinear relationship exists between biological age and time.     

Double minutes replicate once during S phase of the cell cycle

Double minutes (dm) characteristically exhibit greater numerical heterogeneity among tumor cells than do chromosomes. The biological basis of this heterogeneity was studied in human carcinoma cell line S 18. Pulse labeling of asynchronous cells with [³H]dThd, continuous labeling of synchronized cells with BrdUrd and prematurely condensed chromosome (PCC) studies of G1 and G2 phase S 18 cells indicate that dm-DNA replicates only once during S phase of the cell cycle. No evidence was found for replication of dm-DNA at G1 phase, G2 phase or mitotis. Cells observed at anaphase show imprecise distribution of dm to daughter cells. These studies suggest numerical heterogeneity of dm results from anomalous mitotic segregation rather than anomalous replication of dmDNA.     

Chronology of chromosome reduplication in Chinese hamster cells incubated at low temperature

Experiments have been carried out on Chinese hamster fibroblasts. Cultures at the log-stage of growth were incubated at 15 or 25° C for 24 hrs. In two groups of experiments the cells were labeled with H³-TdR for 6 hrs at the respective temperature, washed and further incubated at 37° C. In each group of experiments cultures labeled with H³-TdR at 37° C for 20 min were used as a control. It was found: 1. the delay in the onset of cell passage through the mitotic cycle at 37° in cultures exposed to 15 or 25° C was about equal to 1,5-1 hr resp. and cells proceeded through the life cycle without blockages at any phase of the cycle; 2. the patterns of chromosome reproduction during the second half of the S-phase were the same after labeling at 15, 25 and 37° C. — In the third group the cells were labelled with HP-TdR for 10–60 min and 6 hrs resp. at 25° C. The patterns of reproduction of chromosome pairs 1–4 and small metacentrics were found to be the same in cells labeled briefly and those labeled for 6 hrs. After brief labeling asynchronous reduplication of different segments in many chromosomes became evident. It was masked because of heavy labeling after 6 hrs treatment.