Membrane-associated proteins of adriamycin sensitive and resistant murine leukemic P388 cells

We have isolated an 84-fold adriamycin resistant subline, P388/R84, from mouse leukemia P388 cells by serial cultivation in methylcellulose in the presence of increasing drug concentrations. Electrophoresis of detergent soluble fractions of radiolabeled sensitive and resistant cells suggested marked alterations in the protein fractions of 160, 100, 60, 45, and 30 kd. In resistant clones labeled with 125I an increase in 160 and 100 kd proteins was accompanied by concomitant reduction in the 60, 45, and 30 kd proteins. In 35S methionine-labeled resistant cells, similar increases in the 160 and 100 kd components were observed but in contrast to 125I-labeled cells the 30 kd component was also higher. Alterations in surface proteins were confirmed in experiments where the cell extracts were adsorbed to concanavalin A polymers and extracted with 0.26 M methyl-alpha-D-mannopyranoside. Our data confirm earlier reported observations on cell-surface protein changes in cells resistant to anthracyclines and alkaloids.     

Immunologic heterogeneity of dihydrofolate reductase from methotrexate sensitive and resistant L1210 leukemia cells

Dihydrofolate reductase from L1210 leukemia cells which are sensitive and resistant to methotrexate has the same physical and kinetic properties and immunoreactivity with a guinea pig antiserum raised to the enzyme purified from the methotrexate resistant strain. However, a chicken antiserum to dihydrofolate reductase from methotrexate sensitive L1210 cells has greater affinity for the homologous enzyme than for the enzyme from the MTX resistant cells indicating that there is some antigenic difference in these molecules.     

Comparative accumulation of the Hoechst 33258 fluorescent probe in leukemia P388 cells sensitive and resistant to doxorubicin

The authors studied accumulation of the fluorescent probe Hoechst 33258 in leukemia P 388 sensitive (P 388/0) and resistant to doxorubicin (P 388/DOX) cells. It was shown that intensity of fluorescence of the dye increased after binding with nuclear DNA during 25 min for both lines of the cells. Intensity of fluorescence was 40% greater in sensitive than resistant cells. If Triton X-100 was added no difference between two lines of the cell was observed. When doxorubicin was added to the cells with dye, the intensity of fluorescence decreased. It was suggested to use Hoechst 33258 for assessment extent doxorubicin accumulation in nuclei of the cells.     

Isolation and characterization of a murine P388 leukemia line resistant to clofarabine

A murine P388 leukemia line fully resistant to clofarabine was obtained after only two courses of intraperitoneal treatment (three times a day for nine consecutive days). The resistance was stable for at least 13 weeks without treatment. The subline was as sensitive to 5-fluorouracil, methotrexate, cyclophosphamide, cisplatin, melphalan, BCNU, doxorubicin, etoposide, irinotecan, vincristine, and docetaxel as was the parental P388/0 line but was cross-resistant to five antimetabolites [palmO-ara-C, 4'-thio-ara-C, fludarabine phosphate, cladribine, and gemcitabine-all of which require deoxycytidine kinase for activation] and paclitaxel. The subline had less than 1% of the deoxycytidine kinase activity in comparison to P388/0.     

Protein kinase C-gamma is present in adriamycin resistant HL-60 leukemia cells

The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells.     

Localization of dihydrofolate reductase amplified genes in Chinese hamster cells resistant to methotrexate

New sublines of BFFR1 and BFFR3 cells were obtained as a result of prolonged cultivation of Chinese hamster cells of Blld-ii-FAF 28 line (clone 431) in the presence of increasing concentrations of methotrexate (MTX). The lines obtained were resistant to 200 and 300 mcM of MTX, respectively. Amplification of the gene for dihydrofolate reductase (DHFR), similar to normal DHFR gene in restriction patterns, was proved by blot-hybridization of the resistant cells' DNA with 32P-labeled plasmid DHFR-26. Correlation is shown between the extent of gene amplification and resistance of the cell lines. In situ hybridization of the metaphase chromosomes of resistant cells with 3H-DHFR-26 results in preferential binding of the label with the regions of marker chromosomes 2 and 5, containing long, so called differential staining regions which are known to be the places of localization of amplified genes.     

Ultrastructural alterations in plasma membranes from drug-resistant P388 murine leukemia cells

Freeze-fracture studies of daunomycin-sensitive and daunomycin-resistant P388 cell lines, reveal a significant increase in the numerical density of intramembrane particles at both, the protoplasmic and the exoplasmic leaflets of the plasma membrane from the drug-resistant cells. Such change in plasma membrane architecture is not accompanied by overexpression of P-glycoproteins. Furthermore, drug-sensitive cells exhibited an increased number of exo-endocytotic images when compared to drug-resistant cells. Our observations suggest that there are global changes in the structural organization of the plasma membrane, which are related to the acquisition of the cellular drug-resistant phenotype.     

Differences in myristic acid synthesis and in metabolic rate for P388 cells resistant to doxorubicin

Lipids extracted from doxorubicin-resistant murine leukemia cells (P388/ADR) contained greater relative amounts of myristic and palmitoleic acids than lipids from sensitive cells (P388). This was seen in both the phospholipid and neutral lipid fractions under two nutritional conditions. Correspondingly in P388/ADR cells, myristic acid comprised a greater proportion of the products of the fatty acid synthetase system, and acyl-CoA 9-desaturase activity was transiently greater than in P388. Similar alterations in myristic acid synthesis were exhibited by DC3F/AD X, N417/VP-16, and P388/AZQ30U cells but not by CHRC5 or HL60/AR cells. This alterations was independent of alterations in the P180 glycoprotein and might be linked via the myristoylation of proteins to a different mechanism of drug resistance. Doxorubicin-resistant P388/ADR cells also exhibited a much higher rate of oxidative energy production.     

Changes in glutathione-S-transferase activity during induction of resistance of leukemia P 388 and Ehrlich ascitic tumor cells to doxorubicin]

We have studied by uridine short term test the level of resistance of murine leukemia cell lines P 388/Dx and ELD/Dx carcinoma cells with induced resistance to doxorubicin, P 388/Fp + Dx cells with induced resistance to combination of finoptOFF++ and doxorubicin in vivo. It was shown that the level of resistance was 6 fold for P 388/Dx cells, 4.5 fold for ELD/Dx cells and 2 fold for P 388/Fp + Dx cells. It was shown that the P 388/Dx cells and P 388/Fr + Dx cells had a 3.5 and 4.4 fold increase level of glutathione-S-transferase activity than P 388 cells. No increase in the activity of glutathione-S-transferase was detected in ELD/Dx cells. We conclude that increase of cellular glutathione-S-transferase activity is not associated with the development of resistance to doxorubicin.     

Identification of multi-drug resistant cells in sensitive Friend leukemia cells by quantitative videomicrofluorimetry.

The cellular resistance to cytotoxic drugs, particularly to anthracyclines, remains a major problem in cancer chemotherapy. A number of biochemical mechanisms have been described, one of them being a lower accumulation of drugs in resistant cells. The accumulation of Ho33342 in sensitive and resistant Friend leukemia cells was studied by quantitative fluorescence image analysis, a method which allows investigations to be made on living tissues and cells. The intensity of fluorescence is related to the amount of Ho33342 accumulated into the cells and has been found to be more intense in sensitive cells than in resistant ones. Moreover, the retention of this vital dye was inversely related to the degree of resistance in the three resistant cell lines. The addition of verapamil, which is known to reverse resistance to anthracyclines, resulted in an increase of the amount of Ho33342 accumulated in the resistant cells. Ho33342 presents a higher quantum yield than any other anthracyclines, such as adriamycin and can be used as a microfluorimetric probe to identify the resistant cells in a heterogeneous cell population.     

Effects of cis-unsaturated fatty acids on doxorubicin sensitivity in P388/DOX resistant and P388 parental cell lines

It has been reported that several cis-unsaturated fatty acids (c-UFAs) could increase doxorubicin (DOX) accumulation in cancer cells and hence elevate its cytotoxicity. However, some researchers showed that c-UFA pretreatment did not affect its cytotoxicity in special cell lines. It is possible that the different results occurred due to different cellular characteristics. We hypothesized that c-UFA treatment might modulate the activities of some antioxidant enzymes to affect the resistance of cells to DOX. In the present study, we examined how c-UFA pretreatment affected DOX cytotoxicity on mouse leukemia cell line, P388, and its resistant subline, P388/DOX, which we found to have significantly higher glutathione peroxidase (GPx) activity as well as P-glycoprotein (p-gp) overexpression. We chose two c-UFAs, gamma-linolenic acid (GLA) (18:3n-6) and docosahexaenoic acid (DHA) (22:6n-3). Cytotoxicity was measured by MTT (3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion assays. DOX accumulation and p-gp expression were measured by flow cytometry. The activities of catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), and GPx were determined for both cell lines with and without treatment with GLA or DHA. Significant DOX accumulation occurred in both cell lines with GLA or DHA pretreatment, but without any change in p-gp expression in either cell line. Sensitivity to DOX cytotoxicity was improved by GLA or DHA pretreatment in P388/DOX in which only SOD activity was significantly increased, but not in the parental cell line P388 in which both SOD and CAT were significantly increased by the pretreatment. However, combined pretreatment of GLA or DHA with antioxidants, pyrrolidinedithiocarbamate (PDTC) or Vitamin C, could sensitize not only P388/DOX but also P388 cells to DOX. We conclude that the effects of c-UFA pretreatment on the sensitivity of cancer cells to DOX not only depend on the change in drug accumulation but also the change in the levels of antioxidant enzyme activities, and suggest that combined administration of c-UFAs, antioxidants, and DOX may be more effective in treating leukemia.     

Dihydrofolate reductase activity and resistance to aminopterin in various species of Drosophila

Summary The aim of our work was to compare the mechanisms of resistance to aminopterin, inhibitor of the dihydrofolate reductase enzyme, between different Drosophila species and those described for cultured cells. Moreover we compared the systematic species divisions based on morphological traits and those based on a molecular approach. For this purpose, the effect of aminopterin on viability and wing phenotype was studied in different Drosophila species. Dihydrofolate reductase was measured in adult flies. We found an important dihydrofolate reductase activity in the melanogaster sub-group compared to the other species studies. Wing effect was observed only in this sub-group. The effects of aminopterin on the wing phenotype were very similar to the phenotype of rudimentary mutants. Both deplete the pyrimidine pool and it has been shown by the studies of the structural genes of the nucleotide pyrimidine pathway that the wing tissue is very sensitive to every pertubation of this metabolism.The D. ananassae species was found to be fully resistant at the concentrations of the inhibitor tested. No or very little dihydrofolate reductase activity was detected. The binding of the enzyme to the inhibitor was comparable to that found in the Oregon strain of D. melanogaster. The purine and pyrimidine salvage pathways were investigated and the D. ananassae species displayed an important thymidine kinase activity. The D. ananassae flies were sensitive on Sang medium compared to the Oregon flies but were able to use exogenous bases or nucleosides more efficiently. Therefore the mechanism of resistance to aminopterin in Drosophila may be different from those described for methotrexate in mammalian cultured cells, as indicated by the results obtained for D. ananassae.     

Biotransformation of mafosfamide in P388 mice leukemia cells: intracellular 31P-NMR studies

The intracellular transformation of cis-mafosfamide has been studied in P388 mice leukemia cells using 31P-NMR spectroscopy. For this purpose the cells were entrapped in low-gelling-temperature agarose threads. Internal pH of the cells, determined from the position of the intracellular inorganic phosphate, was 7.2. The cell membrane was permeable to 4-hydroxycyclophosphamide and aldophosphamide and less permeable to phosphoramide mustard. 4-Ketocyclophosphamide and carboxyphosphamide signals were not detectable in cells either sensitive or resistant to oxazaphosphorine treatment.     

The expression of P-glycoprotein in leukemia P388 cells with induced doxorubicin resistance]

beta-D-galactose-containing glycoproteins were prepared from cells P-388 leukemia and from P-388 leukemia cells with induced resistance to doxorubicin. It was shown by HPLC method that plasma membranes from resistant cells contain 4-4.5% P-glycoproteins and plasma membranes from sensitive cells contain P-glycoproteins about 10 times lower.     

Dihydrofolate reductase and aminopterin resistance in Pneumococcus

Summary Wild-type pneumococci derived from Avery's strain R36A are sensitive to extracellular concentrations of the folate antimetabolite aminopterin exceeding 1.0x10^-6 M. Three classes of resistant strains are phenotypically distinguishable: amiB-r, amiA-r and amiD-r strains are resistant to low (1.5x10^-6 M), intermediate (0.5–4.0×10^-5 M) and high (4.5x10^-4 M) aminopterin levels respectively. The amiA and amiB regions are weakly linked, but linkage has not been established between either of these loci and the amiD region.Consistent with the maximum resistance conferred by mutations in the amiA locus, dihydrofolate (FH₂) reductase in cell-free extracts (CFE) of amiA-r strains has a two- to six-fold greater affinity for the substrate than dose the enzyme in wild-type CFE (Table 1); FH₂ reductase from amiA-r strains may also have reduced affinity for aminopterin. Specific activity of the enzyme is not affected by mutation in the amiA locus (Table 1) and its affinity for the cofactor (NADPH) is probably unaffected by mutation in this locus (Table 4). Dihydrofolate reductase activity in amiA5 CFE is considerably more thermolabile than that in wild-type CFE (Table 2).The enzyme in CFE of the high resistance strain amiD1 has the same affinity for the substrate, cofactor and antimetabolite as FH₂ reductase in wild-type CFE (Figs. 1–4, 8 and 9; Table 4). However, specific activity of the enzyme in amiD1 CFE is 11-fold higher than that in wild-type CFE (Table 1) and it is much more heat stable (Table 2).Some properties of FH₂ reductase in CFE of the high resistance recombinant strain amiA5amiD1 are intermediate between those in CFE of wild-type and amiD1.Preliminary results suggest that CFE of wild-type and amiA5 contain a factor, which is neither dialyzable nor heat sensitive, that has an inhibitory effect upon activity and stability of FH₂ reductase in amiD1 CFE (Tables 2 and 3).