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In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells.

Four new variants of L1210 cells resistant to endoplasmic reticulum (ER) stressors, tunicamycin (STun), thapsigargin (SThap), bortezomib (SBor), and MG-132 (SMG-132), were developed via an 18-month periodic cultivation in culture medium with a gradual increase in substance concentration. Multidrug resistance was generated for STun (to tunicamycin, bortezomib and MG-132), SThap (to tunicamycin, thapsigargin and MG-132), SBor (to bortezomib and MG-132), and SMG-132 (to bortezomib and MG-132). These cells were compared to the original L1210 cells and another two variants, which expressed P-gp due to induction with vincristine or transfection with the gene encoding P-gp, in terms of the following properties: sensitivity to either vincristine or the ER stressors listed above, proliferative activity, expression of resistance markers and proteins involved in the ER stress response, and proteasome activity. The resistance of the new cell variants to ER stressors was accompanied by a decreased proliferation rate and increased proteasome activity. The most consistent change in protein expression was the elevation of GRP78/BiP at the mRNA and protein levels in all resistant variants of L1210 cells. In conclusion, the mechanisms of resistance to these stressors have certain common features, but there are also specific differences.

Background: Meconopsis horridula Hook (M. horridula) has been used as a traditional Tibetan medicine to relieve heat and pain as well as mobilize static blood, and it is recognized as a good treatment for bruises. This study is the first trial to evaluate the tumor inhibitory activity of M. horridula extract and its underlying mechanism in the hope of providing evidence to support the anticancer function of M. horridula. Methods and Results: M. horridula extract was cytotoxic to L1210 cells in a dose- and time-dependent manner. SEM (scanning electron microscope) observation revealed obvious morphological changes in L1210 cells after M. horridula treatment. Flow cytometry analysis demonstrated that the extract dose-dependently induced early apoptosis. Additional apoptosis parameters, such as alterations in nuclear morphology and DNA damage, were also observed. Furthermore, M. horridula treatment induced G2/M arrest. M. horridula treatment significantly increased reactive oxygen species (ROS) production, suggesting that ROS are a key factor in M. horridula-induced apoptosis. Volatile constituent detection found 15 abundant chemicals in M. horridula, which may contribute to its anticancer effect. Conclusion: In conclusion, M. horridula extract induced L1210 cell apoptosis and inhibited proliferation through G2/M phase arrest, and ROS were involved in the process.

We describe the screening of a set of cryptopleurine derivatives, namely thienoquinolizidine derivatives and (epi-)benzo analogs with bioactive phenanthroquinolizidine alkaloids that induce cytotoxic effects in the mouse lymphocytic leukemia cell line L1210. We used three variants of L1210 cells: i) parental cells (S) negative for P-glycoprotein (P-gp) expression; ii) P-glycoprotein positive cells (R), obtained by selection with vincristine; iii) P-glycoprotein positive cells (T), obtained by stable transfection with a human gene encoding P-glycoprotein. We identified the most effective derivative 11 with a median lethal concentration of ≈13 μM in all three L1210 cell variants. The analysis of the apoptosis/necrosis induced by derivative 11 revealed that cell death was the result of apoptosis with late apoptosis characteristics. Derivative 11 did not induce a strong alteration in the proportion of cells in the G1, S or G2/M phase of the cell cycle, but a strong increase in the number of S, R and T cells in the subG1 phase was detected. These findings indicated that we identified the most effective inducer of cell death, derivative 11, and this derivative effectively induced cell death in S, R and T cells at similar inhibitory concentrations independent of P-gp expression.

Overexpression of P-glycoprotein (P-gp, drug transporter) in neoplastic cells is the most frequently observed molecular cause of multidrug resistance. Here, we show that the overexpression of P-gp in L1210 cells leads to resistance to tunicamycin and benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside (GalNAc-α-O-benzyl). Tunicamycin induces both glycosylation depression and ubiquitination improvement of P-gp. However, the latter is not associated with large increases in molecular mass as evidence for polyubiquitination. Therefore, P-gp continues in maturation to an active membrane efflux pump rather than proteasomal degradation. P-gp-positive L1210 cells contain a higher quantity of ubiquitin associated with cell surface proteins than their P-gp-negative counterparts. Thus, P-gp-positive cells use ubiquitin signaling for correct protein folding to a higher extent than P-gp-negative cells. Elevation of protein ubiquitination after tunicamycin treatment in these cells leads to protein folding rather than protein degradation, resulting at least in the partial lack of cell sensitivity to tunicamycin in L1210 cells after P-gp expression. In contrast to tunicamycin, to understand why P-gp-positive cells are resistant to GalNAc-α-O-benzyl, further research is needed. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp) overexpression is the most frequently observed cause of multidrug resistance in neoplastic cells. In our experiments, P-gp was expressed in L1210 mice leukemia cells (S cells) by selection with vincristine (R cells) or transfection with the gene encoding human P-gp (T cells). Remodeling of cell surface sugars is associated with P-gp expression in L1210 cells as a secondary cellular response. In this study, we monitored the alteration of cell surface saccharides by Sambucus nigra agglutinin (SNA), wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA). Sialic acid is predominantly linked to the surface of S, R and T cells via α-2,6 branched sugars that tightly bind SNA. The presence of sialic acid linked to the cell surface via α-2,3 branched sugars was negligible, and the binding of MAA (recognizing this branch) was much less pronounced than SNA. WGA induced greater cell death than SNA, which was bound to the cell surface and agglutinated all three L1210 cell-variants more effectively than WGA. Thus, the ability of lectins to induce cell death did not correlate with their binding efficiency and agglutination potency. Compared to S cells, P-gp positive R and T cells contain a higher amount of N-acetyl-glucosamine on their cell surface, which is associated with improved WGA binding. Both P-gp positive variants of L1210 cells are strongly resistant to vincristine as P-gp prototypical drug. This resistance could not be altered by liberalization of terminal sialyl residues from the cell surface by sialidase.

The acceleration of drug efflux activity realized by plasma membrane transporters in neoplastic cells, particularly by P-glycoprotein (P-gp, ABCB1 member of the ABC transporter family), represents a frequently observed molecular cause of multidrug resistance (MDR). This multiple resistance represents a real obstacle in the effective chemotherapy of neoplastic diseases. Therefore, identifying cytotoxic substances that are also effective in P-gp overexpressing cells may be useful for the rational design of substances for the treatment of malignancies with developed MDR. Here, we showed that triorganotin derivatives—tributyltin-chloride (TBT-Cl), tributyltin-bromide (TBT-Br), tributyltin-iodide (TBT-I) and tributyltin-isothiocyanate (TBT-NCS) or triphenyltin-chloride (TPT-Cl) and triphenyltin-isothiocyanate (TPT-NCS)—could induce the death of L1210 mice leukemia cells at a submicromolar concentration independently of P-gp overexpression. The median lethal concentration obtained for triorganotin derivatives did not exceed 0.5 µM in the induction of cell death of either P-gp negative or P-gp positive L1210 cells. Apoptosis related to regulatory pathway of Bcl-2 family proteins seems to be the predominant mode of cell death in either P-gp negative or P-gp positive L1210 cells. TBT-Cl and TBT-Br were more efficient with L1210 cells overexpressing P-gp than with their counterpart P-gp negative cells. In contrast, TBT-I and TPT-NCS induced a more pronounced cell death effect on P-gp negative cells than on P-gp positive cells. Triorganotin derivatives did not affect P-gp efflux in native cells measured by calcein retention within the cells. Taken together, we assumed that triorganotin derivatives represent substances suitable for suppressing the viability of P-gp positive malignant cells.

Multidrug resistance (MDR) is a serious obstacle to the effective chemotherapeutic treatment of leukemia. Expression of plasma membrane P-glycoprotein (P-gp), a transporter involved in drug efflux, is the most frequently observed molecular causality of MDR. We observed the coexpression of P-gp and the filament protein nestin in the acute myeloid leukemia (AML) cell lines SKM-1 and MOLM-13 following the induction of P-gp expression using vincristine. Nestin is considered a marker of neural stem cells and neural progenitor cells. The aim of this study was to determine whether there is causal relationship between the expression of P-glycoprotein and the expression of nestin in both of these AML cell lines. The expression of P-gp was induced in SKM-1 cells by selective pressure using vincristine (VCR), mitoxantrone (MTX), azacytidine (AzaC) and lenalidomide (LEN). Whereas the selective pressure of VCR, MTX and AzaC also induced P-gp expression in MOLM-13 cells, LEN was found to be ineffective in this regard. In all cases in which P-gp expression was induced in SKM-1 and MOLM-13 cells, its expression was associated with the induction of nestin mRNA expression and the presence of a 200–220 kDa nestin-immunoreactive protein band in western blots. Silencing P-gp expression using s10418 siRNA (known as the P-gp silencer) was associated with the downregulation of the nestin transcript level, demonstrated using RT-PCR. Nestin mRNA was also observed in two P-gp-positive variants of L1210 cells that were obtained either by selection with VCR or by transfection with a retrovirus encoding human P-gp. Detectable levels of nestin transcripts were not observed in P-gp-negative parental L1210 cells. Taken together, these results indicated that the induction of P-gp expression is causally associated with the expression of nestin in leukemia cells. [ABSTRACT FROM AUTHOR]

The interaction of dimethyl-N-(benzoyl)amidophosphate (HCP) and dimethyl-N-(phenylsulfonyl)amidophosphate (HSP) compounds and C60 fullerene with DNA was studied by use of molecular modeling. It was demonstrated that DNA-HCP complex is more stable as compared to the DNA-HSP one. The ability of C60 fullerene to form a stable complex with minor groove of DNA was shown. Under combined action of 10-5 M C60 fullerene and 2.5 × 10-4 M HCP the viability of leukemia L1210 cells was decreased by 37% in 24 h. The observed cytotoxic effect was intensified in 48 h. HSP compound alone as well as at combined action with C60 fullerene showed no cytotoxic effect. [ABSTRACT FROM AUTHOR]

Overexpression of P-glycoprotein (P-gp, drug transporter) in neoplastic cells is the most frequently observed molecular cause of multidrug resistance. Here, we show that the overexpression of P-gp in L1210 cells leads to resistance to tunicamycin and benzyl 2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAc-α-O-benzyl). Tunicamycin induces both glycosylation depression and ubiquitination improvement of P-gp. However, the latter is not associated with large increases in molecular mass as evidence for polyubiquitination. Therefore, P-gp continues in maturation to an active membrane efflux pump rather than proteasomal degradation. P-gp-positive L1210 cells contain a higher quantity of ubiquitin associated with cell surface proteins than their P-gp-negative counterparts. Thus, P-gp-positive cells use ubiquitin signaling for correct protein folding to a higher extent than P-gp-negative cells. Elevation of protein ubiquitination after tunicamycin treatment in these cells leads to protein folding rather than protein degradation, resulting at least in the partial lack of cell sensitivity to tunicamycin in L1210 cells after P-gp expression. In contrast to tunicamycin, to understand why P-gp-positive cells are resistant to GalNAc-α-O-benzyl, further research is needed.

Background: Meconopsis horridula Hook (M. horridula) has been used as a traditional Tibetan medicine to relieve heat and pain as well as mobilize static blood, and it is recognized as a good treatment for bruises. This study is the first trial to evaluate the tumor inhibitory activity of M. horridula extract and its underlying mechanism in the hope of providing evidence to support the anticancer function of M. horridula. Methods and Results: M. horridula extract was cytotoxic to L1210 cells in a dose- and time-dependent manner. SEM (scanning electron microscope) observation revealed obvious morphological changes in L1210 cells after M. horridula treatment. Flow cytometry analysis demonstrated that the extract dose-dependently induced early apoptosis. Additional apoptosis parameters, such as alterations in nuclear morphology and DNA damage, were also observed. Furthermore, M. horridula treatment induced G2/M arrest. M. horridula treatment significantly increased reactive oxygen species (ROS) production, suggesting that ROS are a key factor in M. horridula-induced apoptosis. Volatile constituent detection found 15 abundant chemicals in M. horridula, which may contribute to its anticancer effect. Conclusion: In conclusion, M. horridula extract induced L1210 cell apoptosis and inhibited proliferation through G2/M phase arrest, and ROS were involved in the process. [ABSTRACT FROM AUTHOR]

Abstract: Sonodynamic therapy (SDT) has shown great potential in target cancer therapy, but it induced cell death modes has not been fully investigated. This study was to examine autophagic and apoptotic responses to protoporphyrin IX (PpIX) mediated SDT in murine leukemia L1210 cells. After SDT, the occurrence of autophagy was identified by morphological observation and biochemical analysis. Meanwhile, the mitochondria dependent apoptosis pathway was examined to participate in SDT induced cell death. The relationship between autophagy and apoptosis was further investigated by applying pharmacological inhibition studies, which indicated that impairment of autophagy enhanced the anti-tumor effect of SDT through induction of apoptosis and necrosis, while caspase inhibition did not affect autophagic vacuoles formation or protect SDT induced cytotoxicity. The findings supported that autophagic vacuoles formed upstream and independently from caspase-dependent cell death. Additionally, the possible mechanism of SDT-induced autophagy was evaluated by measurement of ROS (reactive oxygen species) formation. Result suggested ROS play important role in initiating autophagy, possibly through the sono-damaged mitochondria being enclosed by autophagic vacuoles. All together, these data indicate that autophagy may be cytoprotective in our experimental system, and point to an important insight into how autophagy inhibitors, in combination with SDT may contribute a regimen for cancer therapy. [Copyright &y& Elsevier]

According to the data of laser-correlation spectroscopy of the pristine fullerene C60 water colloid solution used in the study the average hydrodynamic radius of nanoparticles was 50 nm and no agglomeration was observed. With the use of the fluorescent probe 2′,7′-dichlorofluorescein the production of reactive oxygen species (ROS) in rat thymocytes and in human leukemic cells L1210 pretreated with 10-5 M fullerene C60 and irradiated in a diapason 320-600 nm was estimated. Irradiation per se is shown to be followed by slight increase of ROS production. No further increase of fluorescent signal was detected in thymocytes during 3 h of incubation after combined action of C60 and irradiation. In contrast leukemic cells respond to combined action of C60 and irradiation by pronounced intensification of ROS production. This phenomenon is shown to be connected with prooxidant-antioxidant imbalance induced by photoexcited fullerene C60 in L1210 cells, enhancement of the first superoxide dismutase against decreasing of the second glutathione peroxidase steps of antioxidant defense. The prooxidant effect of photoexcited fullerene C60 leading to oxidative stress could be used for elaborating the strategies of targeted oxidative injury of leukemic cells. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Equisetum hyemale has been used as a traditional herbal medicine to treat various diseases such as hypertension, inflammatory diseases, acute stroke, bleeding and cancer. The present study aimed to investigate the anti-proliferative effect and the underlying mechanisms of E.hyemale extract on murine leukemia L1210 cells. Materials and methods: The inhibitory effect of Ehyemale extract on L1210cells was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cell cycle distribution was evaluated with flow cytometry following PI (propidium iodide) staining. Apoptotic cell death was determined by Annexin V–FITC/PI and nuclear DAPI (4′-6-diamidino-2-phenylindole) staining. DNA damage and changes of mitochondrial membrane potential were also detected with flow cytometry analysis. Results: E.hyemale extract exerted significant antiproliferative effects on L1210 cells in a dose- and time-dependent manner. Flow cytometry analysis showed that E.hyemale extract induced cell cycle arrest at G2/M phase in L1210 cells. Phosphatidylserine exposure, chromatin condensation, DNA damage and loss of mitochondrial membrane potential were observed clearly after treatment with Ehyemale extract. Conclusion: The results in this study indicate that E.hyemale extract could inhibit L1210 cell proliferation through inducing G2/M arrest and cell apoptosis. [Copyright &y& Elsevier]

P-glycoprotein (P-gp) overexpression is the most frequently observed cause of multidrug resistance in neoplastic cells. In our experiments, P-gp was expressed in L1210 mice leukemia cells (S cells) by selection with vincristine (R cells) or transfection with the gene encoding human P-gp (T cells). Remodeling of cell surface sugars is associated with P-gp expression in L1210 cells as a secondary cellular response. In this study, we monitored the alteration of cell surface saccharides by Sambucus nigra agglutinin (SNA), wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA). Sialic acid is predominantly linked to the surface of S, R and T cells via a-2,6 branched sugars that tightly bind SNA. The presence of sialic acid linked to the cell surface via a-2,3 branched sugars was negligible, and the binding of MAA (recognizing this branch) was much less pronounced than SNA. WGA induced greater cell death than SNA, which was bound to the cell surface and agglutinated all three L1210 cell-variants more effectively than WGA. Thus, the ability of lectins to induce cell death did not correlate with their binding efficiency and agglutination potency. Compared to S cells, P-gp positive R and T cells contain a higher amount of N-acetyl-glucosamine on their cell surface, which is associated with improved WGA binding. Both P-gp positive variants of L1210 cells are strongly resistant to vincristine as P-gp prototypical drug. This resistance could not be altered by liberalization of terminal sialyl residues from the cell surface by sialidase. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we investigated the effects of 24 chalcone derivatives from 2-naphthylacetophenone toward a lymphoblastic leukemia cell line (L1210). Three compounds, called R7, R13, and R15, presented concentration- and time-dependent cytotoxicity and induced cellular death by apoptosis via mitochondrial injury and oxidative stress. The effects of these compounds appear to occur through different mechanisms because R13 and R7 induced a greater disturbance of mitochondrial potential, and all compounds induced disturbances of cellular ATP content and increased caspase-3 activity before cellular death. These compounds also interfered with antioxidant enzymes activities and GSH content through different mechanisms. [ABSTRACT FROM AUTHOR]

We investigated the antitumor effect of a short-chain polysaccharide (PS) extracted from porcine cartilage both in L1210 leukemic cells in vitro and in mice bearing L1210 ascites tumors. Our results show that treatment with PS resulted in a significant (P < 0.01) inhibition of cell proliferation and caused apoptotic death in L1210 cells. We also found that PS increased the ratio of Bax/Bcl-2 and activated caspase-9 and caspase-3 but not caspase-8. These results point to the activation of the mitochondrial apoptotic pathway in response to the treatment of L1210 cells with PS. Our results suggest that PS has the potential to provide significant natural defence against leukemias and lymphomas. [ABSTRACT FROM AUTHOR]

Abstract: L1210/VCR cell line (R) was obtained by adaptation of the L1210 mouse leukaemia cells (S) to vincristine and showed P-glycoprotein (P-gp) mediated multidrug resistance (MDR). R cells were observed to be more sensitive to high external calcium as parental S. More pronounced calcium uptake was observed for R cells. Moreover, differences in intracellular calcium cell localization between S and R cells were found ultrastructurally following a calcium precipitating cytochemical method. In S cells, calcium precipitates were found to be localized predominantly along the cell surface coat and within mitochondria delineating the cristae. In R cells, precipitates were also found inside nuclei, at the border of heterochromatin clumps, and scattered within the cytoplasm. High extracellular calcium did not influence the P-gp mediated extrusion of calcein/AM as P-gp substrate. These results indicate that calcium enters and consequently damages the MDR cells to a higher extent than parental cells. [Copyright &y& Elsevier]

In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells. [ABSTRACT FROM AUTHOR]

Cultured L1210 murine lymphocytic leukemia cells were used to compare metabolic activation and cytotoxicity of 5-fluorouracil (FU), Ftorafur (FT), and three novel FU-sulfur analogues. These analogues, l-(2′-tetrahydrothienyl)-5-fluorouracil (FUS), l-(2′-tetrahydrothienyl)-5-fluorouracil-l′-oxide (FUSO), and 1-(2′-tetrahydrothienyl)-5-fluorouracil-l′-l′-dioxide (FUSO2), have yet to be fully evaluated for potential therapeutic value based on in vitro cytotoxicity. The role of these FU analogues as prodrugs was evaluated by comparing metabolism of normal pyrimidine pathways and activation by hepatic mixed function oxidases (MFO). Significant differences in biochemical activity and cytotoxicity were measured between FU and FU analogues. FU and FU analogues were cytotoxic to L1210 cells (63-92% growth inhibition of 100 µ M concentrations after 72 hr of incubation). However, at equimolar concentrations cytotoxicity of the FU analogues after MFO activation (56-66% growth inhibition) was greater than FU (47% growth inhibition). Hypoxanthine, a purine precursor, did not significantly alter fluoropyrimidine cytotoxicity with or without MFO. Thymidine and uridine, pyrimidine precursors, reduced FT and FUS cytotoxicities in the presence (27, 40%) and absence (25, 15%) of MFO but did not modify FU, FUSO, or FUSO2 cytotoxicities. [ABSTRACT FROM AUTHOR]

At present, pharmacokinetic aspects in existing in vitro assays for cytotoxic drug screening are considered only insufficiently. Using a new microperfusion assay, we integrated the peak plasma concentration (Cmax), the time of peak concentration (tmax), the absorption rate, and the elimination rate following methotrexate (MTX) treatment of L1210 leukemic cells in vitro. The effects of different MTX concentration courses with constant exposure doses (area under the concentration-time curve) were checked by combining the microperfusion assay with the clonogenic assay in agar-containing glass capillaries. We found that the concentration profiles obtained in the ultrafiltration-flat chamber (an essential part of the microperfusion system) can be described by the Bateman function. Thus, the flat chamber might be comprehended as a one-compartmental system with first-order absorption kinetics. We found that the colony-inhibition kinetics of L1210 cells obviously depended on the MTX exposure profile. Continuous cell-growth inhibition was obtained by one concentration profile that offered a compromise between all pharmacokinetic parameters. Our results correlated with the known pharmacodynamic activities of MTX and showed the relevance of different concentration courses to the cytotoxic effect of the drug. We suppose that the growth-inhibition kinetics of unknown, potential anticancer drugs can be also interpreted in similar ways. [ABSTRACT FROM AUTHOR]

An L1210 cell line (JT-1), which can grow in medium supplemented with 1 nm folate, has been isolated. These cells exhibit a slower growth rate than folate-replete parental cells and have a lower ability to transport folate or methotrexate via the reduced folate transport system. Measurements at nanomolar concentrations of folate revealed that the adapted cells have acquired a high-affinity folate-binding protein. Binding to this component at 37°C was rapid and reached a maximum value after 30 min which corresponded in amount to 0.23±0.3 pmol/mg protein, and excess unlabeled folate added 30 min subsequent to the [H]folate led to a rapid release of the bound substrate. Radioactivity bound to or released from the cells after 30 min at 37°C remained as unmetabolized folic acid. Binding was also rapid at 0°C but uptake at the plateau was only one-half the value obtained at 37°C. Half-maximal saturation of the binding component ( K) occurred at a folate concentration of 0.065 nm at pH 7.4, while the affinity for folate decreased 30-fold when the pH was reduced to 6.2 ( K=2.0 nm). 5-Methyltetrahydrofolate was also bound by this component ( K=13 nm at pH 7.4) but with a much lower affinity than for folate, while progressively weaker interactions were observed with 5-formyltetrahydrofolate ( K=45 nm) and methotrexate ( K=325 nm). When the same adaptation procedure was performed with limiting amounts of 5-formyltetrahydrofolate, two additional cell lines, JT-2 and JT-3, were isolated which expressed elevated levels of the folate-binding protein. The binding activity of the latter cells was 0.46 and 1.4 pmol/mg protein, respectively. When the level of binding protein was compared in cells grown at different concentrations of folate, an increase in medium folate from 1 to 500 nm caused a sevenfold reduction in binding activity in the JT-3 cell line, while these same growth conditions had no effect on binding by the other cells. These results indicate that L1210 cells adapted to low concentrations of folate or 5-formyltetrahydrofolate contain elevated levels of a high-affinity binding protein and that this protein is able to mediate the intracellular accumulation of folate compounds. L1210 cells thus appear to have two potential uptake routes for folate compounds, the previously characterized anion-exchange system and a second route mediated by a high-affinity binding protein. An additional low-affinity, high-capacity transport system for folate that had been proposed previously was not observed under a variety of experimental conditions in either the adapted or parental cells. [ABSTRACT FROM AUTHOR]

o-Phthalate is actively transported into L1210 cells and the primary route for cell entry is the same transport system which mediates the influx of methotrexate and other folate compounds. The identity of the influx route has been established by the following observations: (A) Phthalate influx is competitively inhibited by methotrexate and the inhibition constant (Ki) is comparable to the Kt for half-maximal influx of methotrexate: (B) Various anions inhibit the influx of phthalate and methotrexate with comparable Ki values; (C) The influx of phthalate and methotrexate both fluctuate in parallel with changes in the anionic composition of the external medium; and (D) A specific covalent inhibitor of the methotrexate transport system (NHS-methotrexate) also blocks the transport of phthalate. In contrast, the efflux of phthalate does not occur via the methotrexate influx carrier, but rather by two separate processes which can be distinguished by their sensitivities to bromosulfophthalein. Efflux via the bromosulfophthalein-sensitive route constitutes 75% of total efflux and is enhanced by glucose and inhibited by oligomycin. The inability of phthalate to exit via the methotrexate influx carrier is due to competing intracellular anions which prevent phthalate from interacting with the methotrexate binding site at the inner membrane surface. [ABSTRACT FROM AUTHOR]

Measurements of methotrexate transport in L1210 cells in the presence and absence of D-glucose reveal that both influx and efflux are depressed in the absence of D-glucose, whereas the steady-state accumulation of drug is enhanced. The reason for the increase in steady state is that the relative decline in efflux is greater than the decline in influx. Analysis of the concentration dependence of steady-state methotrexate accumulation in D-glucose-deprived cells indicates a linear relationship consistent with a one-carrier active transport model. Similar data in nondeprived cells is highly nonlinear and strongly supports the postulate that under physiological conditions influx and efflux of methotrexate are mediated by separate carrier systems. These results indicate that the efflux system, preferentially transporting methotrexate under normal conditions, cannot operate in the absence of D-glucose, whereas the influx system is only partially inhibited under conditions of glucose deprivation. [ABSTRACT FROM AUTHOR]

The transport of [3H] 1,L 5-formyltetrahydrofolate, [3H] folic acid, and [3H]methotrexate by L1210 cell plasma membrane vesicles exhibited multicompartmental behavior. Two separate vesicular compartments (parallel relationship) of approximately equal volume were revealed during measurements of influx and efflux. Flux in one compartment was rapid, saturable, highly temperature-sensitive, and inhibited by pCMBS. Flux in the other compartment exhibited all of the characteristics of passive diffusion. These results imply that our plasma membrane vesicle preparations consist of a mixture of two functional species. Transport of folate into one of these species occurs by passive diffusion alone, whereas transport into the other kind of vesicle occurs by both passive diffusion and carrier-facilitated transport. [ABSTRACT FROM AUTHOR]

The photodynamic effects of a new photosensitizer, meso-tetra(hydroxyphenyl) chlorin (m-THPC), and Photofrin were evaluated and compared in in vitro and in vivo experiments. For in vitro assays, the influence of sensitizer doses and incubation time on the toxicity and phototoxicity of murine leukaemic cells (L1210) were studied. For in vivo assays, the influence of sensitizer and laser doses on the growth of a human tumour (HT29) originated from a human colorectal adenocarcinoma (grafted subcutaneously in mice) were studied. For in vitro assays, LD50 was reached at 1 μg ml for Photofrin and 0.88 μg ml for m-THPC after 2-h incubation on leukaemic cells irradiated at 514 nm with an energy density of 25 J cm. When incubation time was increased, m-THPC-induced photoxicity increased more than Photofrin photoxicity. In vivo, at an energy density of 40 J cm, m-THPC (0.25, 0.5 mg kg) irradiated at 650 nm was more potent than Photofrin (5 mg kg) irradiated at 632 nm but was also more phototoxic to mice. Laser doses seemed to have less influence than m-THPC doses on tumour growth as the tumour growth index was 12 mm at a laser dose of 10 J cm and 13 mm at 40 J cm (21.4 mm for control, p<0.01). It is likely that the greater efficiency of m-THPC was due in part to a higher quantum yield ofO for m-THPC than for Photofrin and to better light transmission at 650 nm in living tissues than at 630 nm. However, microscopic m-THPC distribution throughout tumour sites has to be precise. [ABSTRACT FROM AUTHOR]

The effect of a combined treatment with shock waves generated by a lithotripter and Adriamycin or cisplatin was examined in cells that acutely survived exposure to shock waves and proliferated afterwards. Batches of 2 x 10 cells were exposed to the respective drug for 50 min or for 50 min plus 72 h. During the 50-min drug exposure 500 shock waves were applied at 25 kV. The growth as a percentage of the control was determined after 72 h by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cells treated with shock waves alone showed a growth inhibition as compared to control cells. For a 50-min drug exposure with Adriamycin the dose enhancement ratio did not exceed 1.3. For a 50-min drug exposure with cisplatin at concentrations of 0.5 μg/ml and 5.0 μg/ml, growth (as a percentage of the control) after combined treatment was significantly reduced as compared to cisplatin treatment alone; the dose enhancement ratio was 3.2 at 50% growth compared to the control. This indicates that shock waves can increase the susceptibility of L1210 cells to cisplatin. For a 50-min plus 72-h drug exposure no effect of an additional treatment with shock waves, as compared to chemotherapy alone, could be observed. [ABSTRACT FROM AUTHOR]

The polyamines are cell constituents essential for growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in the polyamine biosynthetic pathway. Methylglyoxal bis(guanylhydrazone) (MGBG) is an anti-leukemic agent with a strong inhibitory effect against AdoMetDC. However, the lack of specificity limits the usefulness of MGBG. In the present report we have used an analog of MGBG, diethylglyoxal bis(guanylhydrazone) (DEGBG), with a much greater specificity and potency against AdoMetDC, to investigate the effects of AdoMetDC inhibition on cell proliferation and polyamine metabolism in mouse L1210 leukemia cells. DEGBG was shown to effectively inhibit AdoMetDC activity in exponentially growing L1210 cells. The inhibition of AdoMetDC was reflected in a marked decrease in the cellular concentrations of spermidine and spermine. The concentration of putrescine, on the other hand, was greatly increased. Treatment with DEGBG resulted in a compensatory increase in the synthesis of AdoMetDC demonstrating an efficient feedback control. Cells seeded in the presece of DEGBG ceased to grow after a lag period of 1-2 days, indicating that the cells contained an excess of polyamines which were sufficient for one or two cell cycles in the absence of polyamine synthesis. The present results indicate that analogs of MGBG, having a greater specificity against AdoMetDC, might be valuable for studies concerning polyamines and cell proliferation. [ABSTRACT FROM AUTHOR]