Hematopoiesis, myeloid leukemia and growth factors.
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Cell Line↗
Biomedical subjects
I F Pryme
Publications and source records attributed to I F Pryme.
Protein factors in extracts of ribosomes isolated from L-929 cells during various phases of the cell cycle.
Centrifugal elutriation has been utilized in order to separate cultures of L-929 fibroblasts into subpopulations containing cells at different stages of the cell cycle. The subpopulations were characterized by Coulter counter volume determination, [3H]thymidine label into DNA and flow cytometry. When a population of early G1 cells was returned to roller culture it was shown to progress through the cell cycle in a synchronous manner. Ribosomal factor extracts were prepared from cells at various phases during the cell cycle. The amounts of protein in the extracts varied greatly, being lowest in early G1 phase and showing a peak during S phase. Polyacrylamide gel electrophoresis demonstrated that there were differences in the protein species present in the extracts. Some proteins were present in the same amounts throughout the cell cycle, whereas others appeared to show a form of cyclical behaviour.
Animals↗
A growth-promoting ribosome extract (GPRE) from mouse L-929 cells stimulates growth of the HL-60 human promyelocytic leukemia cell line.
An extract termed growth-promoting ribosome extract (GPRE), isolated from mouse L-929 cells stimulates growth of HL-60 human promyelocytic leukemia cells. The stimulation first becomes apparent at 72 h when the cells start to enter the quiescent state. The inhibition of protein synthesis by the addition of cycloheximide to L-929 cells before ribosomal extracts were prepared did not alter the stimulatory effect of GPRE. When GPRE was added together with 20% fetal calf serum to cultures of quiescent HL-60 cells, growth was stimulated to the extent that the generation time was reduced by approximately 9 h to 32.4 h. GPRE alone was unable to stimulate the quiescent cells. The growth stimulatory effect was not restricted to one cell generation but was a characteristic of at least the following two cell cycles. GPRE extract from L-cells synchronized by centrifugal elutriation was most efficient when isolated from cells in early G1 phase, while extract from S phase cells had virtually no effect. It is tentatively suggested that the factor belongs to the competence/progression group of growth factors.
Animals↗
The nuclear associated endoplasmic reticulum and membrane biogenesis in MPC-11 cells.
The incorporation of 3H-glucosamine, 3H-choline and 14C-fucose into subcellular fractions of MPC-11 cells was studied. After a 20 min period of labelling with both 3H-glucosamine and 3H-choline, greatest incorporation was observed in nuclear-associated endoplasmic reticulum (NER). 14C-fucose, however, was incorporated to a greater extent in endoplasmic reticulum (ER) membranes. Pulse-chase experiments with 3H-glucosamine showed a loss of radioactivity from NER and a simultaneous increase in the ER fraction. In comparison to NER, ER membranes were poorly labeled with 3H-glucosamine after a 20 min pulse. Following a 2 h incubation there was a 12 fold increase in radioactivity in ER membranes in comparison to a 1.2 fold increase in NER. There were no individual differences between subfractions of ER membranes with respect to 3H-glucosamine content after the pulse, or following the 2 h incubation. The results indicate that the NER is a major, early site of the synthesis of 3H-glucosamine labeled membrane glycoproteins, and that these proteins migrate into other ER membranes early after their synthesis.
Cell Line↗
Cytochalasin B binding and actin content of endoplasmic reticulum subfractions isolated from L-929 cells.
Heavy rough (HR) endoplasmic reticulum (ER) membranes and a dense fraction of light rough (LR) membranes (LR I) of L-929 cells bind H-cytochalasin B extremely poorly in comparison to smooth (S) membranes and a fraction of LR membranes of low density (LR II). The LR and S subfractions of ER are apparently heterogeneous membrane populations with respect to cytochalasin B binding. The separation of proteins in HR and LR subfractions by electrophoresis followed by immunoblotting with monoclonal antibodies against actin showed that actin was not present in the former membranes while there were large amounts in the LR subfraction. It is concluded that membranes in the LR II fraction of ER are associated with actin-containing microfilaments of the cytoskeleton, but that HR membranes are not.
Actins↗
Compartmentation of the rough endoplasmic reticulum.
It has become evident during recent years that a wide variety of proteins are synthesized on membrane-bound polysomes, very many of which are not ultimately secreted from the cell. The majority of proteins appear to go through some form of post-translational modification before the final appearance of an 'active' product, and in some cases the polypeptide chain may be modified before the completed protein molecule is released from the ribosome. This then raises the question concerning the possibility of the organization of the rough endoplasmic reticulum into individual domains, or compartments, each of which may have the responsibility of performing definite and well defined functions. During recent years the behaviour of two subfractions of the rough endoplasmic reticulum in a variety of cell types and under a variety of conditions has been studied in order to gain insight into a possible compartmentation of this organelle. Throughout the studies disruption of cells has been performed by nitrogen cavitation. This technique was chosen in order to provide conditions of homogenization which were extremely reproducible since shearing forces, mechanical damage and the effects of local heating were eliminated. Endoplasmic reticulum (ER) membranes isolated from the post-mitochondrial supernatant have been separated into subfractions by centrifugation on discontinuous sucrose gradients. By virtue of their high density imparted by the association of ribosomes, rough ER (RER) membranes penetrate 1.4 M sucrose accumulating above either 2.0 M sucrose (light rough -LR membranes) or a cushion of 2.3 M sucrose (heavy rough -HR membranes). Smooth (S) membranes, which are virtually devoid of ribosomes, collect above 1.4 M sucrose. The HR, LR and S subfractions in MPC-11 cells differ in a number of respects: RNA/protein and RNA/phospholipid ratios, polysome profiles and marker enzymes. When cells were homogenized in buffer containing 25 mM KCl then all three ER subfractions were observed, however, when the buffer contained 100 mM KCl then only the LR and S subfractions were observed in gradients, radioactivity equivalent to that in the HR fraction was not recovered in the other two subfractions. Four times as many light chain immunoglobulin polypeptides were found associated with polysomes of HR membranes compared to LR membranes. The nuclear associated ER (NER), though very active in protein synthesis, was only 20% as active in the synthesis of light chain as the combined LR/HR fraction. Studies with MPC-11 cells showed that the relative amounts of the three ER subfractions were related to the phase of the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
The importance of salt concentration during nitrogen cavitation of MPC-11 cells for the isolation of heavy rough endoplasmic reticulum membranes.
When MPC-11 cells are disrupted by nitrogen cavitation in the presence of buffer containing 25-40 mM KCl then endoplasmic reticulum membranes can be separated into three subfractions by sucrose density gradient centrifugation: heavy rough (HR), light rough (LR) and smooth (S) membranes. An increase in the salt concentration of the buffer to 50 mM or above results in the occurrence of only the LR and S membranes in gradients. However, when cells equilibrated at high pressure in the bomb in 100 mM KCl buffer were expelled into a diluting buffer such that the final buffer concentration was reduced to 25 mM KCl upon cell disruption, then appreciable amounts of HR membranes are observed in sucrose gradients. The results would suggest that salt concentrations above 25-40 mM KCl stabilize the interaction between HR membranes and the cytoskeleton to such a degree that these membranes are pelleted at low speed together with the nuclei. The yields of LR and S membranes are apparently not affected to any significant degree by altered salt concentration.
Animals↗
In vitro transformation and tumor promotion studies of styrene and styrene oxide.
The carcinogenic properties of styrene and styrene oxide were investigated using C3H/10T1/2C18 cells as a test system. In vitro transformation was not observed for either of the two chemicals; however, styrene oxide at three different concentrations enhanced the morphological transformation in the two-stage transformation assay. 0.1, 1 and 10 microM styrene oxide added twice weekly resulted in 32.4, 26.8 and 31.4 per cent of the dishes with one or more type III foci. Styrene and styrene oxide were only slightly toxic to the cells at the concentrations used. Styrene oxide did not affect the growth rate of the C3H/10T1/2 cells at 10 microM. However, 100 microM styrene oxide added to logarithmically growing cells caused a significant decrease in growth rate within 24 to 48 h. The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate inhibited DNA synthesis approximately 60% 8 h after initiation of treatment. When styrene oxide at concentrations up to 100 microM was tested in a similar experiment, however, no significant effect was observed. Total RNA synthesis increased by 70% 1.5 h after initiation of treatment at 1 microM styrene oxide; this effect was not seen after 24 h. Styrene oxide at concentrations of 1 and 0.1 microM stimulated the incorporation of [3H]choline into cells by approximately 20% during a 2 h incubation, the major site of incorporation being the nuclear-associated endoplasmic reticulum.
Animals↗
Chilling followed by incubation at 37 degrees causes a flow of 3H-choline labeled phospholipids from nuclear-associated endoplasmic reticulum to other endomembranes of L-cells.
The exposure of L-cells to a period of 15 min incubation in ice followed by a return to normal culture conditions caused distinct alterations in the distribution pattern of 3H-choline incorporation in phospholipids in subcellular fractions. The amount of radioactivity appearing in the nuclei and nuclear-associated endoplasmic reticulum decreased while that in the mitochondrial and endoplasmic reticulum membrane fractions increased, suggesting a precursor-product relationship. These changes appeared to occur in a linear fashion. Furthermore, the increase in radioactivity in individual endoplasmic reticulum subfractions differed in that label increased fivefold in light rough membranes but only about twofold in the HR and S subfractions.
Animals↗
Altered distribution profiles of endoplasmic reticulum subfractions after incubation of Krebs II ascites cells with different concentrations of cytochalasin B.
Information on the interaction between endoplasmic reticulum (ER) membranes and components of the skeletal network of the cell was gained by treating cells with the antimicrofilament agent cytochalasin B prior to cell disruption by nitrogen cavitation. Treatment of Krebs II ascites cells with cytochalasin B (5-10 micrograms ml-1) resulted in an increased yield of three ER membrane subfractions--heavy rough (HR), light rough (LR) and smooth (S) membranes, as judged by 3H-choline incorporation in gradient fractions following discontinuous sucrose gradient centrifugation. The major increase was observed in the HR fraction. These results indicate that the actual yield of the respective ER membrane subfractions after cell disruption is dependent on the degree of direct and/or indirect interaction between individual ER membranes and actin containing filaments of the cytoskeleton in the intact cell.
Actins↗
Light rough endoplasmic reticulum membranes re-appear before heavy rough membranes after feeding starved MPC-11 cells.
In 48 hr starved MPC-11 cells smooth (S) endoplasmic reticulum (ER) membranes accounted for more than 80% of the total ER membrane fraction. After 2 1/2 hr feeding the amount of S membranes was reduced to below 55% while light rough (LR) membranes had increased from about 16% of the total to 42%. The percentage of heavy rough (HR) membranes only showed a minor increase during the incubation period. The results show that following an accumulation of S membranes in starved cells the LR membranes 're-appear' more rapidly than the HR membranes. This difference in behaviour provides further support to the concept that LR and HR membranes represent distinct domains of the ER system.
Animals↗
The nuclear-associated endoplasmic reticulum is an early target for the action of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate in C3H/10T1/2 fibroblasts.
12-O-Tetradecanoyl-phorbol-13-acetate (TPA) had a dual effect on the cellular membranes of C3H/10T1/2 cells in that it caused both a stimulation of [3H]choline incorporation and an enhancement of the solubilization of choline from prelabelled cells. Subfractionation studies showed that the release of [3H]choline occurred almost exclusively from nuclear-associated endoplasmic reticulum. The release was dependent on the presence of Mg2+ and Ca2+, indicating an enzyme-mediated reaction. In vivo, TPA stimulated the incorporation of [3H]choline into all subcellular fractions. The data indicate that the nuclear-associated endoplasmic reticulum represents an early target for TPA action.
Animals↗
Time-dependent alteration in endoplasmic reticulum membrane profiles during in vitro incubation of Krebs II ascites cells.
Krebs II ascites cells were harvested from the mouse peritoneum 6-8 days after inoculation and incubated in vitro in roller suspension culture for up to 22 hr. Within 2 hr of incubation a large portion of the cells entered S phase as judged by the incorporation of 3H-thymidine into DNA. The incorporation of radioactive precursors into phospholipid, protein and RNA increased rapidly during in vitro incubation indicating a high degree of macromolecular synthesis. The rates of incorporation were maximal within 4 hr of incubation. When cells labeled with 3H-choline were disrupted by nitrogen cavitation and endoplasmic reticulum (ER) membranes analyzed for subfractions on discontinuous sucrose gradients, it was observed that only small amounts of radioactivity could be detected in the HR region after 1/2 hr incubation while 63.2% of total radioactivity in ER membranes appeared in the LR fraction. Between 8-18 hr there was a considerable increase in the amount of HR membranes. Of the total radioactivity in ER membranes that in the HR fraction increased from 16.9% to 53.9% during this period. There were only small changes in the amounts of radioactivity in LR and S membranes between 8 and 18 hr. The results suggest a time-dependent appearance of ER membrane subfractions during a 22 hr period of in vitro incubation of Krebs II ascites cells.
Animals↗
The phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate and stimulation of 3H-choline incorporation into endoplasmic reticulum membranes and other subcellular fractions of Krebs II ascites cells during in vitro incubation.
After transfer of Krebs II ascites cells from the mouse peritoneum to suspension culture addition of the phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) causes an early stimulation of 3H-choline incorporation into phosphatidylcholine (PC). Choline transport into the treated cells, however, was unaffected. Within 30 min of TPA treatment 3H-choline incorporation was almost 300% above the control level. During a 5 hr period of suspension culture the overall patterns of 3H-choline incorporation were similar in TPA-treated and control cultures though the rate was greatly accentuated by the presence of the phorbol ester. Incubation of cells with cycloheximide prior to incubation with TPA did not result in an inhibition of the TPA-directed 3H-choline incorporation. After 3 hr incubation with TPA there were large increases in radioactivity in all subcellular fractions. At 20 hr, however, the values were not far from those of the control. During the first 3 hr of incubation with TPA the incorporation of 3H-choline into light rough (LR) and smooth (S) membranes was stimulated to levels of 400% and 320% respectively above control values. At later times the profiles of radioactivity in membrane subfractions in TPA-treated and control cultures were similar. The results illustrate an early effect of TPA on PC biosynthesis in Krebs II ascites cells while at later times of incubation the stimulatory effect was virtually abolished.
Animals↗
The specific appearance of a 65,000 dalton protein in the nuclear associated endoplasmic reticulum of Krebs II ascites cells early after treatment with the phorbol ester TPA.
The protein compositions of subcellular fractions prepared from untreated and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-treated Krebs II ascites cells were examined by SDS-polyacrylamide gel electrophoresis. After 6 h of TPA treatment a 65,000 dalton protein in the nuclear associated endoplasmic reticulum showed a 240% increase when compared with the untreated control cells. The amount of this protein was similar in both untreated and TPA-treated cells up to 4 h of in vitro incubation. Though present in all other subcellular fractions, the amount of the 65,000 dalton protein was the same in both untreated and TPA-treated cells. Small changes were also observed in the amounts of other proteins in the nuclear associated endoplasmic reticulum in TPA-treated cells. The possibility was tested that the increased amount of the protein may have represented increased ornithine decarboxylase activity in response to TPA treatment. The time dependent appearance of the enzyme in untreated and TPA-treated cells, however, and the behaviour and subcellular localization of the 65,000 dalton protein ruled out the possibility that this protein and ornithine decarboxylase are one and the same molecule.
Animals↗
Ultrastructure and polysome content of the microsomal subfractions of mouse plasmacytoma cells.
The endoplasmic reticulum (ER) of MPC-11 cells released as vesicles upon cell disruption by nitrogen cavitation was separated from the bulk of mitochondria, lysosomes and plasma membranes by a low speed centrifugation. The ER membranes were fractionated on discontinuous sucrose gradients into heavy rough (HR), light rough (LR) and smooth (S) membranes. The morphology of subcellular fractions was studied by electron microscopy and the ER membranes were shown to be virtually free of contaminating organelles. The S fraction was easily distinguishable because of the lack or ribosomes but there were no apparent morphological differences between the HR and LR fractions. Of total activity in the microsomal subfractions, 70% of the UDPase and 67% of the 5'-nucleotidase activity was associated with the S fraction. Polysomes were present in the HR, LR and nuclear-associated ER fractions but not in the S fraction. The HR and LR fractions did not appear to be contaminated to any great extent with free polysomes. RNA/protein and RNA/phospholipid ratios of the HR fraction were higher than those of the LR fraction, indicating a greater density of ribosomes in the former fraction. These ratios were much lower in the S fraction reflecting the low ribosome content.
5'-Nucleotidase↗
Variation in microsomal subfractions obtained from normal animal tissues and transformed cells following cell disruption by nitrogen cavitation.
Microsomal membranes were obtained from MPC-11 cells, L-cells, Krebs II ascites cells and various normal animal tissues following cell disruption by nitrogen cavitation. Membrane preparations were applied to discontinuous sucrose gradients designed to separate three fractions--heavy rough (HR), light rough (LR) and smooth (S) microsomes. In each of the transformed cell lines all three fractions were found whilst in the normal tissues tested the HR fraction was absent. Of the normal tissues liver and pancreas were rich in both LR and S microsomes, the presence of large amounts of LR indicating a rich protein synthesizing activity on membrane-bound polysomes. Kidney also contained appreciable LR but much less than both liver and pancreas. Both heart and lung contained virtually only S microsomal material--a reflection of low protein synthetic activity on membrane-bound polysomes. Attempts to promote the appearance of the HR fraction in liver, kidney and pancreas by incubation in tissue culture medium, or, in the case of pancreas, by cholecystokinin/pancreozymin/secretin stimulation both in vivo and in vitro were unsuccessful.
Animals↗
Comparison of the rate of aminoacylation of tRNA isolated from NMRI mouse liver with tRNA isolated from Krebs II ascites or mouse plasmacytoma cells.
A comparison of the initial rates of aminoacylation of tRNAs isolated from different sources for 17 amino acids was performed. tRNA was isolated from NMRI mouse liver (tRNA L) and from Krebs II ascites tumors (tRNA Asc), and aminoacyl-tRNA synthetases were prepared from the latter cells. The aminoacylation of tRNA Asc was 31-88% slower than the charging of tRNA L. In similar studies, tRNA from a mouse plasmacytoma tumor (tRNA Mt) and from suspension cultured cells of the same cell line (tRNA M) were compared to tRNA L in the aminoacylation reaction catalysed by synthetases isolated from tumor or suspension cultured cells. About half of the tRNAs (Mt or M) for the 17 amino acids tested differed in charging rate when compared to tRNA L, but the differences were not as great as those observed in the experiments where tRNA Asc and tRNA L were compared.
Amino Acyl-tRNA Synthetases↗