Effect of n-butyrate on cell cycle progression and in situ chromatin structure of L1210 cells

In the presence of 1–5 mM n-butyrate, murine leukemic L1210 cells cease proliferation and become arrested in the G1A compartment of the G1 phase. Cells in this compartment, in comparison with the remaining cells of the G1 phase (G1B), are characterized by low RNA content and more condensed chromatin. During unperturbed growth the cell residence times in G1A are of indeterminate duration (exponentially distributed); the half-time of L1210 cell residence in G1A is about 1.4 h. The effect of n-butyrate in arresting cells in G1A was concentration-dependent. However, the sensitivity of L1210 cells to this drug was markedly enhanced when cells were treated for longer than one generation (12 h). Cells arrested in G1A remained viable and when n-butyrate was removed, after a lag period, they resumed progression through the cycle.The effect of n-butyrate on cell progression through various parts of the cycle was studied in a stathmokinetic experiment. The rate of cell entrance into mitosis was decreased by 30, 60 and 110%, in the presence of 1, 2.5 and 5 mM n-butyrate respectively, thus indicating a slowdown in cell progression through G2 and S. The duration of G2 was prolonged by 20, 70 and 140% at 1, 2.5 and 5 mM n-butyrate respectively. The half-time of cell residence in G1A was increased by as much as 1.5-, 6.3- and 15.6-fold by 1, 2.5 and 5 mM n-butyrate. Progression through late G1 (G1B) was not affected at 1 mM, and could not be estimated at higher drug concentrations. The effects on cell cycle progression were evident 1 h after addition of n-butyrate.DNA in situ in nuclei of n-butyrate-treated cells had lowered (by 2–8 °C) stability to thermal denaturation and increased (by 15%) accessibility to DNase I. The decrease in DNA stability to heat was more pronounced when permealized cells were heated in the presence of 1 mM MgCl₂ rather than EDTA. DNA in situ in the nuclei of n-butyrate-treated cells also showed decreased sensitivity to acid-induced denaturation. Changes in chromatin were seen in all cells, regardless of cell cycle phase, within the first hours after addition of n-butyrate. Mitotic cells, however, reacted to n-butyrate more rapidly than interphase cells. The observed changes in L1210 cells are most likely a consequence of histone modifications (acetylation of inner histones, dephosphorylation of histone H1) induced by n-butyrate.     

Effect of methotrexate on the leukemic cell cycle in mouse ascitic leukemia L1210]

In cells of L1210 ascite leukemia cells, methotrexate inhibited H3-thymidine incorporation, blocked shortly (during 4 hours) the G1 leads to S transition, and did not affect cells in G2-phase or in the late S phase. Almost half a cell population was degenerated and cells in S- and G1-phases were affected in equal proportion. This may suggest that methotrexate is not S-phase specific for cells of leukemia L1210. A simultaneous administration of vinblastine increases the antitumour effect of methotrexate. Cells in G2-phase constitute, presumably, a significant proportion of cells recovered after methotrexate administration. A comparison of the data obtained with literature evidence shows that in the sensitive (leukemia L1210) and resistant (acute mieloid leukemia of man) forms of leukemia, methotrexate affects cells that are in S-phase, whereas cells being in G1-phase are affected only when the sensitive tumours are treated.     

Galactosyltransferase activity and cell growth: uridine diphosphate (UDP)galactose inhibition of murine leukemic L1210 cells

UDPgalactose inhibits the growth of mouse leukemic L1210 cells. In calf serum supplemented Dulbecco's medium (CS-DMEM), 1.2 mM UDPgalactose (UDPgal) inhibited cell growth by 50% (IC50), and 5 mM UDPgalactose inhibited cell growth by 92%. Other nucleotide sugars as well as galactose, glucose, and galactose-1-phosphate had little or no effect on cell growth. Uridine nucleotides, which inhibit galactosyltransferase activity, protected L1210 cells from the growth inhibitory effect of UDPgalactose when both were added simultaneously to culture media. Unlike mouse 3T12 cells, in which no inhibition of cell growth was observed with heat-inactivated calf serum (HICS)-DMEM, 5 mM UDPgalactose inhibited L1210 cell growth in HICS-DMEM to the same degree as that observed in CS-DMEM. In contrast to 3T12 cells, L1210 cells secrete significant galactosyltransferase activity into the media. Complete inhibition of 3T12 cell growth by UDPgal was observed if HICS-DMEM medium was first conditioned by L1210 cells for 48 hours. No difference in cell growth or [3H]thymidine uptake was detected after 6 hours of exposure to UDPgalactose, but both were significantly decreased at 24 and 48 hours. Flow cytometric analysis of UDPgalactose effects on L1210 cells revealed no differences in the distribution of cells in G1, S, or G2-M of the cell cycle after 6 hours of incubation, but after 16 hours of UDPgalactose treatment, L1210 cells were arrested in early S phase. These cells were completely viable and morphologically similar to control L1210 cells. Normal growth was resumed when UDPgal was removed. The data suggest that UDPgalactose inhibition of cell growth requires extracellular galactosyltransferase activity and that the effect is mediated via the cell membrane.     

Caffeine reverses the cytotoxic and cell kinetic effects of Novantrone (mitoxantrone)

Caffeine is known to potentiate the cytotoxicity of a variety of DNA damaging agents presumably by reducing the ability of the cells to repair potentially lethal lesions. However, in the present study we observe that 5 mM caffeine reverses the cell kinetic and cytotoxic effects of the intercalating drug Novantrone (mitoxantrone) on L1210, HL-60 and CHO cells. Novantrone alone, at a concentration of 20-30 ng/ml, given to cultures for 1 h, inhibits cell growth by about 50% and causes cells to accumulate in S and G2 phase and to enter a higher DNA ploidy level. Treatment of these cell lines with 5 mM caffeine alone for 1 h has a minimal effect on cell proliferation; suppression of cell growth varies from 5 to 10%. Exposure of cells to Novantrone for 1 h in the presence of caffeine results in a significant reduction of the Novantrone effects; the cell growth rate is partially restored (e.g. caffeine reduces suppression of L1210 cell growth from 48 to 83% of control) and in each of the cell lines studied, the Novantrone-induced cell accumulation in S and G2 is abolished. Combined treatment with caffeine and Novantrone also increases the clonogenicity of CHO cells 8.5 times over that seen in cultures treated with Novantrone alone. In contrast to the combined treatment with caffeine + Novantrone, pretreatment of cells with caffeine provides no protection. Likewise, post-treatment with caffeine provides little reversal of growth inhibition and G2 cell accumulation, especially if the post-treatment is delayed in time. The present data, in conjunction with evidence in the literature that caffeine protects cells against the cytotoxic effects of doxorubicin, suggest that caffeine may play a more general role in protecting cells against planar aromatic molecules such as intercalating agents.     

Structural alterations of peripheral nerve monogalactosylceramides during development and Wallerian degeneration

Reaction products of selenite with thiols were tested for an inhibitory effect on amino acid incorporation in a cell-free system derived from rat liver and on protein synthesis in intact P815 and L1210 cells. In the cell-free system maximum inhibition, up to 96%, was reached at about 10 microM selenium. In intact cells inhibitory effect varied depending on which reaction product or cell line was used. Maximum inhibition was obtained after 30 min of incubation with selenium concentrations ranging from 0.25 microM to over 7 microM. Selenite itself also inhibited protein synthesis of L1210 cells, but only after 90 min of incubation and starting at selenium concentrations of 2 microM. Inhibition of protein synthesis in intact cells was followed by cell death. Pre-incubation of the reaction products of a monothiol (2-propanethiol) and of a vicinal dithiol (2,3-dimercapto-1-propanol) in culture medium showed a rapid decrease of the inhibitory capability of the product from the monothiol, but not of the product from the dithiol. The results indicate that selenite and a thiol react to form products which have differential toxic effects to cells in vitro.     

Selective inhibition by bis(2-chloroethyl)methylamine (nitrogen mustard) of the Na+/K+/Cl- cotransporter of murine L1210 leukemia cells

Incubation of L1210 murine leukemia cells in vitro with 10 microM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener 86Rb+ to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of 86Rb+ influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359-367) as being mediated by a Na+/K+/Cl- cotransporter. Inhibition by 10 microM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of 86Rb+ influx, mediated by Na+/K+-ATPase. After 3 h of incubation with 10 microM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the [3H]TPMP+, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K+ influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumor activity, did not inhibit 86Rb+ influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 +/- 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na+/K+/Cl- cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.     

The effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on cell growth, cell cycle, induction of DNA fragmentation, and activity of caspase 3 in murine leukemia L1210 cells and fibroblast NIH-3T3 cells

Quinazolines are multitarget agents, which have broad spectrum of biological activity, and some of them are now in cancer clinical testing. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects on cancer cell lines HeLa and B16. Quinazoline, at micromolar concentrations, induced morphological changes and necrosis of B16 cells, and at nanomolar concentrations it produced changes of F-actin cytoskeleton. It did not cause changes in the cell cycle, did not induce apoptotic cell death in B16 cells, did not have a mutagenic effect, and did not even behave as a typical intercalating agent. Little significant reduction of tumor volume in intramuscular transplanted B16 cells was observed. The aim of the present study was to examine the cytotoxic effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on murine leukemia L1210 cells and fibroblast NIH-3T3 cells. Induction of cell morphology and cell cycle changes, induction of apoptosis and caspase 3 activity were studied. Quinazoline acted cytotoxically on both cell lines. The sensitivity of leukemia L1210 cells to the quinazoline was higher than that of fibroblast NIH-3T3. The IC(100) was 12 microM for L1210 cells and 24 microM for NIH-3T3 cells. No effect of quinazoline on the cell cycle profile of L1210 and NIH-3T3 was detected, however, quinazoline induced an increase of the sub-G(0) cell fraction, apoptotic DNA fragmentation, and apoptotic morphological changes at a concentration of 12 microM. This quinazoline concentration induced caspase 3 activity. Our results demonstrated that induction of apoptotic cell death via activation of caspase 3 contributed to the cytotoxic effects of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline in murine leukemia L1210 cells.     

Isolation of tumor disturbing factor on the proliferation of tumor cells in human serum

The classified sediment with ethanol from sera of nude mice and humans showed a disturbing effect on L1210 cells in vitro and a lifesaving effect on L1210 cell-bearing mice in vivo. This factor was purified more than 2300-fold to a specific activity of approximately 1 X 10(5) U/mg by ethanol classified precipitation, Sephadex G-200 gel filtration, DEAE cellulose ion exchange chromatography with Nacl and pH gradient aqueous solution, and preparative polyacrylamide gel electrophoresis.     

Characterisation of a Na+/K+/Cl- cotransporter in alkylating agent-sensitive L1210 murine leukemia cells

The mode of influx of 86Rb+, a K+ congener, to exponentially proliferating L1210 murine leukemia cells, incubated in a Krebs-Ringer buffer, has been characterised. The influx was composed of a ouabain-sensitive fraction (approx. 40%), a loop diuretic-sensitive fraction (approx. 40%) and a fraction which was insensitive to both types of inhibitor (approx. 15%). The fraction of ouabain-insensitive 86Rb+ influx, which was fully inhibited by furosemide (1 mM) or bumetanide (100 microM), was completely inhibited when Cl- was completely substituted by nitrate or gluconate ions, but was slightly (29 +/- 12%) stimulated if the Cl- was substituted by Br-. The substitution of Na+ by Li+, choline or tetramethylammonium ions inhibited the loop diuretic-sensitive fraction of 86Rb+ uptake. These results suggested that a component of 86Rb+ influx to L1210 cells was mediated via a Na+/K+/Cl- cotransporter. 86Rb+ efflux from L1210 cells which had been equilibrated with 86Rb+ and incubated in the presence or absence of 1 mM ouabain, was insensitive to the loop diuretics. Additionally, efflux rates were found to be independent of the external concentration of K+, suggesting that efflux was not mediated by K+-K+ exchange. The initial rate of 86Rb+ influx to L1210 cells in the plateau phase of growth was reduced to 44% of that of exponentially dividing cells, the reduction being accounted for by significant decreases in both ouabain- and loop diuretic-sensitive influx; these cells were reduced in volume compared to cells in the exponential phase of cell growth. In cells which had been deprived of serum for 18 h, and which showed an increase of the proportion of cells in the G1 phase of the cell cycle, the addition of serum stimulated an immediate increase in the furosemide-sensitive component of 86Rb+ influx. Diuretic-sensitive 86Rb+ influx was not altered by the incubation of the cells with 100 microM dibutyryl cyclic AMP, but was inhibited by 10 microM of the cross-linking agent nitrogen mustard (bis(2-chloro-ethyl)methylamine, HN2).     

Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.     

Suppression of in vitro primary immune response by L1210 cells and their culture supernatant: Evidence for cytotoxic effects

L1210 cells and their culture supernatants were found to inhibit the generation of PFC in the in vitro primary immune response of spleen cells to SRBC. As few as 1% of L1210 cells and 1% of culture fluid were inhibitory. Inhibition of DNA or protein synthesis of L1210 cells did not abolish their immunosuppressive activity, excluding exhaustion of culture medium as a possible mechanism of inhibition of PFC. Heating of the supernatant completely abrogated the suppressive effect and resulted in a marked increase of PFC. Daily evaluation of cell viability in the cultures revealed that, in the presence of L1210 and supernatants, the fraction of surviving cells is markedly reduced. We conclude that a direct cytotoxic effect on splenic lymphocytes and macrophages is the predominant immunosuppressive mechanism of L1210 cells and their culture supernatants.     

Purification and properties of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase from L1210 cells

Serine hydroxymethyltransferase and the trifunctional enzyme C1-tetrahydrofolate synthase have been purified to near homogeneity from L1210 cells. Kinetic constants (Km and kcat) have been determined for both folate and non-folate substrates. The effect of increasing glutamate chain length on affinity and catalytic efficiency were determined for the four activities. The studies show that the structural and catalytic properties of the two L1210 enzymes are very similar to the corresponding enzymes purified from rabbit liver. Antibodies to both rabbit serine hydroxymethyltransferase and C1-tetrahydrofolate synthase cross-react with the corresponding L1210 enzymes. The intracellular concentration of active sites of serine hydroxymethyltransferase and C1-tetrahydrofolate synthase in L1210 cells are both 9 microM. The combined concentration of these two enzymes exceeds the previously reported concentration of 10 microM for total intracellular folates. A network thermodynamic computer model of one carbon metabolism (Seither, R. L., Trent, D. F., Mikulecky, D. C., Rape, T. J., and Goldman, I. D. (1989) J. Biol. Chem. 264, 17016-17023) suggests that complete inhibition of cytosolic serine hydroxymethyltransferase would neither significantly decrease the rates of biosynthesis of purines and thymidylate nor significantly alter the rate of interconversion of tetrahydrofolate cofactors to dihydrofolate with subsequent inhibition of dihydrofolate reductase.     

The different inhibiting effect of cholera toxin on two leukemia cell lines does not correlate with their toxin binding capacity

The murine leukemia cell lines L1210 and WEHI-3B show a very different sensitivity to the cholera toxin (CT).Thein vitro growth of L1210 is completely inhibited by 10^–8 M CT, while WEHI-3B growth shows the same inhibition at 10^–11 M.The analysis of membrane ganglioside pattern of the two cell lines shows that in L1210 cells the major component is the GM1a ganglioside while the monosialoganglioside fraction from WEHI-3B is entirely composed of gangliosides of the b series among which GM1b is the more represented. The total cholera toxin binding capacity of the ganglioside extract from L1210 cells is more than hundred fold higher than that of WEHI-3B and this difference is also confirmed by the number of CT receptors/cell and by the binding of FITC-B subunit of CT on the cells. These surprising data are in conflict with the poor sensitivity to CT evidenced by L1210 compared to WEHI-3B cells.In order to clarify this discrepancy we investigated the cAMP accumulation, the cell viability and the clonogenicity of these two leukemia cell lines following the treatment with CT and forskolin (FRSK).The treatment of WEHI-3B cells with CT induces a dramatic increase of intracellular cAMP which highly correlates with cell death and the decrease of clonogenicity and this result is partially obtained by the treatment with FRSK, L1210 cells do not evidence significant cAMP accumulation neither with CT nor with FRSK treatment.These data suggest that the different inhibiting effect of CT on WEHI-3B and L1210 cells does not correlate with their different pattern of gangliosides and the related toxin binding capacity. Further they indicate that the growth inhibition of WEHI-3B cells is closely related with a cAMP-dependent cell killing mechanism, while the inhibition of L1210 growth (produced by high concentration of CT) is mediated by a cAMP independent mechanism.     

Effect of plasma concentrations of uridine on pyrimidine biosynthesis in cultured L1210 cells

The concentration of uridine in the media of cultured L1210 cells was maintained within the concentration range found in plasma (1 to 10 microM) to determine if such concentrations are sufficient to satisfy the pyrimidine requirements of a population of dividing cells and to determine if cells utilize de novo and/or salvage pathways when exposed to plasma concentrations of uridine. When cells were incubated in the presence of N-(phosphonacetyl)-L-aspartate to block de novo biosynthesis, plasma concentrations of uridine maintained normal cell growth. De novo pyrimidine biosynthesis, as determined by [14C]sodium bicarbonate incorporation into uracil nucleotides, was affected by the low concentrations of uridine found in the plasma. Below 1 microM uridine, de novo biosynthesis was not affected; between 3 and 5 microM uridine, de novo biosynthesis was inhibited by approximately 50%; and above 12 microM uridine, de novo biosynthesis was inhibited by greater than 95%. Inhibition of de novo biosynthesis correlated with an increase in the uracil nucleotide pool. The de novo pathway was much more sensitive to the uracil nucleotide pool size than was the salvage pathway, such that when de novo biosynthesis was inhibited by greater than 95% the uracil nucleotide pool continued to expand and the cells continued to take up [14C]uridine. Thus, the pyrimidine requirements of cultured L1210 cells can be met by concentrations of uridine found in the plasma and, when exposed to such physiologic concentrations, L1210 cells decrease their dependency on de novo biosynthesis and utilize their salvage pathway. Circulating uridine, therefore, may be of physiologic importance and could be an important determinant in anti-pyrimidine chemotherapy.     

Development of host-dependent high-grade tumor-specific immunity through a novel mechanism triggered by the Lyt-2+ tumor-specific T cell clone (K7L) that induces temporal growth of L1210 leukemia-K7L-variant

When a murine leukemia L1210-specific Lyt-2+ T cell clone, K7L, was injected i.p. into CD2F1 mice together with L1210, the normal growth of L1210 in the peritoneal cavity of the mice at the early stage (days 0 to 5) was strongly inhibited, but L1210 grew progressively at the middle-stage (days 5 to 10), and then was rejected at the late stage (days 10 to 20). The mice thus survived for long times (more than 60 days), whereas the normal control injected with L1210 alone died within 14 days. The L1210 that grew at the middle stage in mice initially inoculated with L1210 together with K7L was a K7L-insensitive (K7L-) variant. All of eight tumor clones established from L1210-K7L- by limiting dilution was insensitive to the antitumor activity of K7L, and this property of tumor clones was stable after repeated in vitro passage. The initial depression of the L1210 growth by K7L followed by growth and rejection of the variant L1210-K7L- by the host T cell activity was then found to prepare a strong, long-lasting (more than 3 mo) immunity to protect mice against the high-dose (10(7) cells per mouse) challenge of original L1210. Corresponding to this result, definite tumor (L1210)-specific cytotoxic T lymphocyte (CTL) activity against both variant and original L1210 targets was developed by antigen (L1210) restimulation in the culture of spleen cells from these mice, but was not increased to a detectable level before L1210-K7L- variant started to grow. It was suggested that the 1210-K7L- variant and the original L1210 should have the common tumor-specific antigen that was independent of the K7L-reactive antigen, and that original L1210, whose growth was retarded by K7L, primed the host with the common antigen to be enormously boosted by the subsequently growing L1210-K7L- variant.     

Isolation and characterization of an L1210 cell line retaining the sodium-dependent carrier cif as its sole nucleoside transport activity

Nucleoside permeation across mammalian cell membranes is complex with at least four distinct transporters known. Two of these (es and ei) are equilibrative (facilitated diffusion) carriers that have been studied is considerable detail. The other two (cif and cit) are concentrative, Na(+)-dependent carriers. A major obstacle to the characterization of the latter two mechanisms has been the lack of suitable model systems expressing only a single nucleoside transport activity. The present study describes the isolation of a cell line that has cif as its sole nucleoside transporter. L1210/MC5-1 cells, which have es and cif transport activity, were mutagenized and plated in soft agar containing two cytotoxic nucleosides (tubercidin (7-deazaadenosine) and cytosine arabinoside) that are substrates for es but not cif. A clonal line (L1210/MA-27.1) was isolated which retained the capacity for Na(+)-dependent [3H]formycin B transport but was unable to transport [3H]thymidine, a substrate for es but not cif. Failure of the mutant to transport thymidine was also demonstrated by the inability of thymidine (with adenine as a purine source) to rescue these cells from methotrexate toxicity. Furthermore, the mutant lacked nitrobenzylthioinosine (NBMPR) binding activity (an integral part of the es transporter) as demonstrated by reversible NBMPR binding and photoaffinity labeling with [3H]NBMPR. Loss of es transport activity was also demonstrated by the failure of NBMPR to affect the toxicity of 2-chlorodeoxyadenosine (IC50 approximately 30 nM) in L1210/MA27.1 cells. In contrast, NBMPR decreased the IC50 for 2-chlorodeoxyadenosine from 100 to 30 nM in the parental L1210/MC5-1 cell line. These results are consistent with the mechanism of NBMPR potentiation of 2-chlorodeoxyadenosine toxicity in L1210 cells being a blockade of efflux via es while the nucleoside is pumped into the cells by the concentrative cif carrier.     

Calcium ion-dependent proliferation of L1210 cells in culture

Maximum growth of L1210 cells in culture required the presence of free extracellular calcium ions. Reducing the free extracellular calcium ion concentration with EGTA served to decrease the growth rate of the cells. The decrease in cell growth was not due to cell death but rather due to the "pile-up" of the L1210 cells in the GO/Gl phase of the cell cycle. With the readdition of excess calcium ions, there was a lag period of 3 to 6 hours before the L1210 cells initiated DNA synthesis or transited from the G0/G1 phase to S-phase. Cells enriched for S and G2/M phase by elutriation and which were incubated in EGTA-containing culture medium, continued through the cell cycle and were blocked in GO/Gl. These data indicate that the proliferation of L1210 cells in culture requires a calcium ion-dependent process to allow movement from the G0/G1 to S-phase of the cell cycle.     

Phosphorylation-dephosphorylation of retinoblastoma protein not necessary for passage through the mammalian cell division cycle

Phosphorylation of the retinoblastoma protein (Rb) during the G1-phase of the mammalian cell division cycle is currently believed to be a controlling element regulating the passage of cells into S-phase. We find, however, that the suspension-grown cell lines U937, L1210, and MOLT-4 contain exclusively hyperphosphorylated Rb. Furthermore, when adherent NIH3T3 cells are grown at very low densities to avoid overgrowth and contact inhibition, they also contain only hyperphosphorylated Rb. NIH3T3 cells exhibit hypophosphorylation when the cells are grown at moderate to high cell densities. We propose that cultures of adherent cells such as NIH3T3, when grown to moderate cell densities, are made up of two populations of cells: (a) cells that are relatively isolated and therefore growing exponentially without contact inhibition, and (b) cells that are growth-inhibited by local cell density or contact inhibition. The common observation in adherent cell lines, that Rb is both hyper- and hypophosphorylated in the G1-phase and only hyperphosphorylated in the S- and G2-phases, is explained by the effects of cell density and contact inhibition. Thus, phosphorylation-dephosphorylation of Rb protein during the G1 phase is not a necessary process during the NIH3T3, L1210, MOLT-4, and U937 division cycles. We propose that phosphorylation-dephosphorylation of Rb is independent of the division cycle and is primarily determined by growth conditions throughout the division cycle.