Fluorescence anisotropy of cell membranes of doxorubicin-sensitive and -resistant rodent tumoral cells.

We have studied the plasma membrane fluidity of rat C6 glioblastoma cells and simian virus 40-transformed mouse liver cells in culture that had been rendered resistant to doxorubicin. This was done by the evaluation of fluorescence anisotropy of two probes; diphenylhexatriene was used on membrane microsomal fractions, and trimethylammonium-diphenylhexatriene was used on whole cell suspensions as a plasma membrane-specific probe since it does not enter the cells. A higher degree of membrane fluidity was exhibited with both techniques by doxorubicin-resistant glioblastoma cells as compared to the doxorubicin-sensitive strain, but in the transformed liver cells no such alteration was seen in the physical properties of their plasma membranes. A higher degree of acyl group unsaturation was noticed in the glioblastoma cells but not in the transformed liver cells upon acquisition of doxorubicin resistance. A similar simultaneous increase in acyl group unsaturation and membrane fluidity can be obtained easily by growing the sensitive cells with a medium supplemented with exogenous polyunsaturated fatty acids. This alteration does not modify the sensitivity of the cells to doxorubicin. We conclude from our work that the increase in membrane fluidity, which is frequently associated with drug resistance, is neither necessary nor sufficient for the expression of the resistance. The reason for a link between cell resistance to doxorubicin and plasma membrane fluidity remains to be found.

Fatty acid composition transport and metabolism in doxorubicin-sensitive and-resistant rat glioblastoma cells.

We have studied the lipid composition and the acyl group composition, transport, and metabolism of doxorubicin-sensitive and -resistant rat glioblastoma cells in monolayer cultures (C6 clone). No difference in lipid composition was evidenced; the acyl group composition was, in contrast, highly modified in resistant cells, and these modifications appeared progressively during the acquisition of the resistance. Resistant cells were characterized by a decrease of n-9 eicosatrienoic acid and by a 2-3-fold increase of the proportions of the polyunsaturated fatty acids of the n-6 and n-3 families, especially arachidonic acid and n-3 docosahexaenoic acid. These differences were probably due to a 2-fold increase of the uptake of fatty acids by resistant cells as compared to sensitive cells, this increase allowing the suppression of an essential fatty acid deficiency. Only small changes in the transformations of 16 and 18-carbon atoms' fatty acids to higher analogues were evidenced. A small reduction of the desaturation of stearic acid to oleic acid and of linoleic acid to arachidonic acid was the main characteristic of resistant cells; these differences can be explained as a consequence of the suppression of the essential fatty acid deficiency.

Recognition by human sera of a variable region of the surface glycoprotein of HTLV-I.

The region comprised between the amino acids 175 and 199 of the HTLV-I envelope surface glycoprotein is one of the immunodominant domains of this molecule. In this region, which is well recognized by sera from HTLV-I infected patients, a substitution of the proline at position 192 by a serine has been described in some isolates. Because this mutation could modify the secondary structure of the glycoprotein molecule, we studied the inference of the presence of proline or serine on the recognition of the region 175-199 by human sera. For this, three peptides have been synthetized (a 25-mer 175-199 corresponding to the sequence of the ATK prototype, and two internal 10-mer 190-Pro-199 and 190-Ser-199 having a proline or a serine at position 192) and tested by immunosorbent assay. While most sera reacted with 190-Pro-199 and with 190-Ser-199 synthetic peptides, a differential recognition was observed according to the pathology associated to HTLV-I infection. Moreover sera corresponding to patients infected with a virus harboring a serine at position 192 were found to recognize only the 10-mer with a serine. These data indicates that HTLV-I is subject to antigenic variability.

Characterization of monoclonal antibodies directed against the gp46 of human T-cell leukemia virus type I.

Essential HTLV-I biological functions depend on the structural motives of the surface glycoprotein (gp46). Monoclonal antibodies (mAbs) have been generated in order to identify functional regions of gp46. We obtained three monoclonal antibodies (3F3F10, 4F5F6 and 7G5D8) by immunizing Balb/c mice with beta-propiolactone inactivated HTLV-I producing cells and partially purified gp46. The mAbs are of the IgG 1 subclass. They have been characterized by western blot analysis, reactivity with HTLV-I and HTLV-II producing cells and ELISA binding assays using synthetic peptides. The immunoblot analysis performed with sheets prepared with the virus released by HUT 102 and 2060 cells (an HTLV-I virus producing cell line established in our laboratory) indicate that the three mAbs recognize a 46 kDa product as did the anti -gp46 mAb 0.5 alpha (18). Reactivity of the three mAbs with various cell lines was examined by indirect immunofluorescence assay. The mAb 7G5D8 stained strongly the membrane of all HTLV-I producing cells (MT2, C91/PL, HUT102 and cells of seven lines established in our laboratory and by A. Gessain); uninfected lymphoid cells (HSB-2, MOLT 4, CEM and PHA activated lymphocytes from normal donors) were negative. Interestingly cells of a HTLV-II producing line (344 MO) were positive. The mAbs 3F3F10 and 4F5F6 reacted with the same cells as did 7G5D8 but the fluorescence intensity was much lower than that observed with this later. A long synthetic peptide corresponding to the immunodominant region of the gp46 defined by the amino acids 175-199 and 10-mer peptides overlapping this region were used in an approach to identify the recognized epitope(s). The long 175-199 peptide was recognized by the three mAbs. 3F3F10 and 4F5F6 recognized none of the 10-mer peptides whereas 7G5D8 bound to 186-195 and 182-191 peptides. In addition 7G5D8 did not inhibit either syncytia formation or virus infection. In view of the data concerning the previously described mAbs 0.5 alpha, LAT 27 (5) and KE36-11 (6), our results suggest that the epitope recognized by 7G5D8 is different from those recognized by the former ones. As the 183-191 sequence corresponds to a region in which HTLV-I and HTLV-II harbour six common amino acids and two similar ones, this is consistent with the observation that 7G5D8 stained the HTLV-II producing cells 344 MO as well as all HTLV-I producing ones. Altogether our data support the hypothesis whereby this epitope recognized by 7G5D8 is contained within a sequence defined by amino acids 183-191.

Antibodies directed against a variable and neutralizable region of the HTLV-I envelope surface glycoprotein.

The majority of neutralizing antibodies of HTLV-I are directed against linear epitopes of the envelope surface glycoprotein (gp46) in the immunodominant region 175-199. Although gp46 presents a remarkable degree of conservation, the substitution of the proline at position 192 by a serine is described for 10 isolates among the 54 sequenced ones. This amino acid substitution is known to induce an important change in the orientation of the exposed residues of this region and has drastic consequences on the immunogenicity of the neutralizable epitopes located in this region. We developed monoclonal antibodies directed against epitopes located in this region containing a proline or a serine at position 192. The six monoclonal antibodies obtained recognize the gp46 at the surface of living HTLV-I producing cells, two of them are specific of a 190-197 epitope with a serine at position 192. This demonstrates that the antigenicity of this epitope differs depending on the presence of a proline or a serine at position 192. Altogether, these results demonstrate that the immunodominant neutralizable region 175-199 is antigenically variable.

Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review.

Daunorubicin and doxorubicin, two antibiotics belonging to the anthracycline group, are widely used in human cancer chemotherapy. Their activity has been attributed mainly to their intercalation between the base pairs of native DNA. Complex formation between daunorubicin or doxorubicin with polydeoxyribonucleotides and DNAs of various base composition or chromatins has been investigated by numerous techniques. Many authors have tried to correlate biological and therapeutic activities with the affinity of the drugs for DNA or some specific sequences of DNA. In vivo these anthracycline drugs cause DNA damage such as fragmentation and single-strand breaks. The mechanism of action of anthracyclines involves the inhibition of RNA and DNA syntheses. There exists two limiting factors in the use of anthracyclines as antitumoral agents: a chronic or acute cardiotoxicity and a spontaneous or acquired resistance. In both cases, there is probably an action at the membrane level. It has to be noted that daunorubicin and doxorubicin have a particular affinity for phospholipids and that the development of resistance is linked to some membrane alterations.

Deoxyribonucleic acid of Cancer pagurus. IV. Elution behaviour on hydroxyapatite chromatographic column.

Fractionation of native DNA on hydroxyapatite columns depends, when flat and continuous gradients are used, on the base composition, GC-rich fractions being eluted in the first fractions. Crab satellite DNA behaves abnormally : the first eluted fractions are enriched in poly d(A-T).d(A-T) instead of GC as usual. It amy be suggested that these differences in the behaviour could be attributed to the fact that the secondary structure of crab DNA satellite is different from the secondary structure of the main DNA component.

Establishment of HTLV-I-infected cell lines from French, Guianese and West Indian patients and isolation of a proviral clone producing viral particles.

Human T-cell leukemia virus (HTLV-I) induces adult T cell leukemia/lymphoma (ATL) and a chronic neurological disease named either tropical spastic paraparesis (TSP) or HTLV-I associated myelopathy (HAM). We report here the establishment and characterization of eight HTLV-I-infected lymphoid cell lines derived either from patients with TSP (5) or from asymptomatic carriers (1). Southern blot analysis of T cell beta chain gene rearrangements indicates that all cell lines are composed of clonal populations. The same type of analysis performed with HTLV-I-specific probes showed that they harbor 1 to 5 copies of full length proviruses often associated with deleted proviruses with a restriction map for BamHI, HindIII, PstI and SacI restriction enzymes resembling those of HTLV-I previously isolated from Japan and Caribbean area. One of the cell lines, 2060, derived from a TSP patient was shown to express a relative large amount of virus easily transmissible to fresh peripheral and cord blood lymphocytes. The full length proviral genome contained in this cell line was cloned and used in transient expression experiments. We showed that the cloned provirus was able to direct the synthesis of the major structural viral proteins, the protease and the tax and rex regulatory proteins. The structural viral proteins could be assembled into free particles detected in the culture medium of transfected cells. Although the infectivity of these viral particles remains to be determined, this new clone can be employed to examine the cell types in which this TSP-derived provirus directs viral protein synthesis and eventually replicates. It should also prove of value in studies on the early cellular events induced by viral products.

Sequence variations in the amino- and carboxy-terminal parts of the surface envelope glycoprotein of HTLV type 1 induce specific neutralizing antibodies.

The surface envelope glycoprotein gp46 of the human T cell leukemia virus type 1 elicits a strong immune response. Its protective role against HTLV-1 infection in animal models is well established, suggesting that recombinant envelope glycoproteins or synthetic peptides could be used as an effective vaccine. However, reports have indicated that some variations in envelope sequences may induce incomplete cross-neutralization between HTLV-1 strains. To identify amino acid changes that might be involved in induction of specific neutralizing antibodies, we studied sera from three patients (2085, 2555, and 2709) infected by HTLV-1 with surface glycoprotein gp46 harboring variations in amino acid sequence at positions 39, 72, 265, and 290. Inhibition of syncytia induced by parental, chimeric, or point-mutated envelope proteins indicated that sera 2555 and 2709 primarily recognized neutralizable epitopes located in N- and C-terminal parts of the gp46 glycoprotein. Amino acids changes at positions 39, 265, and 290 greatly impaired recognition of neutralizing epitopes recognized by these two sera. These results demonstrate that amino acid changes in envelope glycoprotein gp46 can induce strain-specific neutralizing antibodies in some patients. On the other hand, the neutralizing activity of serum 2085 was not affected by amino acid changes at positions 39, 265, and 290, suggesting that the neutralizing antibodies present in this serum were directed against epitopes located in other parts of the molecule, possibly those located in the central domain of the molecule, which has the same amino acid sequence in the three viruses.

Influence of amino acid substitutions on antigenicity of immunodominant regions of the HTLV type I envelope surface gylcoprotein: a study using monoclonal antibodies raised against relevant peptides.

By the use of sera of human T cell leukemia virus type I (HTVL-I)-infected individuals it was shown that amino acid substitutions at positions 192 (proline to serine) and 250 (serine to proline) in major immunodominant regions (175-199 and 239-261) of the surface envelope glycoprotein (gp46) of the virus may influence the humoral response. Since human sera are polyclonal in nature, one cannot readily discriminate between an immunoglobulin-specific recognition and multiple bindings of diverse antibodies. To overcome this difficulty we generated murine monoclonal antibodies to synthetic peptides mimicking all or portions of these gp46 regions. The reactivity of some of these antibodies to synthetic peptides harboring (or not harboring) the preceding amino acid substitutions at position 192 or 250, to denatured gp46 by Western blotting, and to live (variously substituted) HTLV-I-infected cells, combined with blocking experiments with various peptides, allow us to conclude that the major epitopes (positions 183-191, 190-197, 190-199, and 246-252) in the two immunodominant regions may elicit different antibody responses according to their sequences. It is worth noting that in a reporter gene inhibition assay, it was found that a neutralizing monoclonal antibody (MF1), the epitope for which is located between residues 190 and 197, had a high level of activity when cells (2060) harboring a gp46 with proline at position 192 were used and had no activity toward cells (1010) with a serine at this position. Therefore our results establish that certain amino acid substitutions of gp46 may drastically affect the antigenicity of the molecule and the biological activity of the antibodies elicited.

Immunogenicity of variable regions of the surface envelope glycoprotein of HTLV type I and identification of new major epitopes in the 239-261 region.

The reactivity of sera of 96 individuals infected with human T-cell leukemia virus type I (HTLV-I) was tested against various synthetic peptides corresponding to the gp46 immunodominant antigenic domains: residues 86-107, 175-199, and 239-261. The frequency of reactive sera was higher for 175-199 (93%) than for 239-261 (78%) or 86-107 (24%) with some variations in geographical regions and in diseases. The region 239-261 was extensively analyzed and five (linear or conformational) epitopes were found. The reactivity of sera toward functional or immunodominant domains may depend on the sequence of the infecting virus, and the role of three frequent substitutions (asparagine by tyrosine, proline by serine, and serine by proline or leucine at positions 93, 192, and 250 respectively) was established. Finally, the role of the genetic background of the host may condition the humoral immune response as individuals infected by HTLV-Is harboring the same predicted gp46 peptide sequence may recognize one, several, or all regions examined.

Pharmacological and molecular characterization of intrinsic and acquired doxorubicin resistance in murine tumor cell lines.

We have studied the pharmacological parameters of doxorubicin resistance in three lines of murine cells selected by long-term culture in the presence of this drug or vincristine. A line originating from rat hepatoma spontaneously presented an intrinsic doxorubicin resistance as compared to the other lines, originating from a rat glioblastoma and from simian-virus-40-transformed mouse hepatocytes. This intrinsic resistance, as well as the doxorubicin resistance exhibited by the vincristine-selected glioblastoma variant, could be entirely attribute to decreased drug accumulation due to drug efflux. In contrast, the doxorubicin-selected variants of the three lines exhibited an intracellular tolerance to this drug. Despite a reduction in drug accumulation when exposed to the same amount of doxorubicin, they accumulated 6-12 times more doxorubicin than wild lines when submitted to equitoxic exposures. Verapamil could restore in these lines the doxorubicin accumulation observed in sensitive lines but could not restore doxorubicin cytotoxicity. Quantitative evaluation of P-glycoprotein expression by Western blotting with the C219 antibody indicated that the wild hepatoma line overexpressed P-glycoprotein by a factor of 5 in comparison with the other wild lines, and that the vincristine-selected glioblastoma variant overexpressed this protein almost as much as the doxorubicin-selected variants. These observations favor the existence of P-glycoprotein-independent mechanisms of doxorubicin resistance, which are added to the classical multidrug-resistant phenotype in doxorubicin-selected highly resistant variant cell lines.

Metabolism of daunorubicin in sensitive and resistant Ehrlich ascites tumor cells. Determination by high pressure liquid chromatography.

The intracellular metabolism of daunorubicin (DNR) has been studied in sensitive and resistant Ehrlich ascites tumor (EAT) cells. The subcellular localization of metabolites has been followed by normal-phase and reverse-phase high-pressure liquid chromatography (HPLC). The metabolism of DNR by either sensitive or resistant EAT cells is not significant; unmetabolized DNR is always the main intracellular compound. Daunorubicinol (DOL) accounts for less than 5% after 24 h and an unidentified product is also observed. This highly apolar compound, having an intrinsic fluorescence one order of magnitude greater than that of DNR is formed in acellular conditions and could be a chemical artifact. DNR and DOL are mainly associated with DNA-containing fractions. No significant differences can be observed in the metabolism of DNR in sensitive and resistant EAT cells.

Chromosomal modifications of a rat glioblastoma cell line during the acquisition and reversal of doxorubicin resistance.

We have studied the cytogenetic alterations occurring during the development and reversal of doxorubicin resistance in a clonal line of rat glioblastoma cells. We have observed during the acquisition of resistance an increase in the modal number of chromosomes, from 42 to 60, and the occurrence, in 90% of the mitoses, or large metacentric markers(s) which were infrequent in the sensitive line. This was associated with a net increase in total DNA amount per cell, from 5.3 to 8.3 pg. During reversal of resistance by 2 years culture without drug of the most resistant line, we observed a rapid decrease of the chromosome number as well as of the DNA content per cell; however, the large metacentric marker(s) were still present in 40% of the mitoses after 9 months of reversal, when the remaining resistance was only 4-fold. In situ hybridization of the chromosomes with a probe for the mdr gene revealed that the average number of stained chromosomes rose from 7% in the sensitive line to 38% in the most resistant line; however, only 9% of the silver grains were detected on the large metacentric markers. We conclude that important chromosome rearrangements occurred during the acquisition of resistance to doxorubicin, leading to a random distribution of the mdr gene in the genome.

Hepatic metabolism of doxorubicin in mice and rats.

We have studied the metabolism of doxorubicin in rat and mouse liver, heart and hepatocytes. Doxorubicinol was present in all cases at very low levels and 7-deoxyaglycones were present only in extraphysiological conditions: no aglycones were found either in fresh livers or in hearts of animals treated with the drug, but they were produced in large amounts when the organs were left at room temperature after the death of the animal. Hepatocytes grown in primary cultures or hepatoma cells grown in continuous lines produced no 7-deoxyaglycones. Freshly isolated hepatocytes synthesized small amounts of 7-deoxyaglycones; however, when these hepatocytes were homogenized prior to incubation, high levels of 7-deoxyaglycones were produced. We conclude that 7-deoxyaglycone formation is possible only in injured tissue and is not, therefore, a normal pathway for doxorubicin.

Cellular pharmacology of doxorubicin in sensitive and resistant rat glioblastoma cells in culture.

We have studied the incorporation and cytotoxicity of doxorubicin in cultured rat C6 glioma cells grown as monolayers. Net incorporation was linear up to high extracellular concentrations of drug (10 micrograms/ml). Cytotoxicity was evaluated both by tritiated thymidine incorporation inhibition and cloning efficiency inhibition. For similar total drug exposures, cytotoxicity was very different according to the exposure time and the exposure dose; incubation with a low dose for a long time was much less cytotoxic than that performed with a high dose for a short period of time. We have obtained several clones of doxorubicin-resistant cells. As compared to the wild strain, these cells were characterized by a larger size, a slower growth, a reduced cloning efficiency and a differential sensitivity of 100-1,000 to doxorubicin. Net incorporation of doxorubicin was 5-fold reduced in these cells, due to an increased efflux of the drug. These cells provide an interesting model of doxorubicin-resistant solid tumor in culture.

Cellular pharmacology of 4'-iodo-4'-deoxydoxorubicin.

We have studied the growth inhibition, DNA synthesis inhibition and cell incorporation of the new anthracycline 4'-iodo-4'-deoxydoxorubicin (4'-iododoxorubicin) and of its 13-dihydroderivative in a model of doxorubicin-sensitive and -resistant rat C6 glioblastoma cells; results were compared to those obtained with doxorubicin and doxorubicinol in the same model. 4'-Iododoxorubicin was 7.5 times more potent than doxorubicin on the wild cell line and 45 times on the doxorubicin-resistant line, indicating that cross-resistance was only partial between the two drugs. Whereas doxorubicinol presented only a very faint cytotoxic activity, 4'-iododoxorubicinol retained the same activity as the parent drug against sensitive cells and a lower activity against resistant cells. DNA synthesis inhibition occurred for much higher doses than growth inhibition in the sensitive cells, but for similar doses in resistant cells. In both cell lines, 4'-iododoxorubicin and its metabolite were incorporated to a higher extent than doxorubicin and doxorubicinol respectively. Incorporation of metabolites was always lower than that of their parent compound. We have studied the metabolism of doxorubicin and 4'-iododoxorubicin by sensitive and resistant cells; only traces (less than 5%) of metabolites were identified in the cells as well as in the culture medium. A new cell line was selected for resistance in the presence of low amounts of 4'-iododoxorubicin. It presented a 6-fold resistance to 4'-iododoxorubicin and an 85-fold resistance to doxorubicin. Doxorubicin incorporation was markedly reduced in this cell line while 4'-iododoxorubicin was incorporated to the same extent as in the sensitive line. Measurements of drug efflux were performed in the three cell lines. No significant difference was exhibited between the efflux of doxorubicin and that of 4'-iododoxorubicin in each cell line; these effluxes were very rapid in the doxorubicin-selected resistant line, slow in the wild line and intermediate in the 4'-iododoxorubicin-selected line.

Deoxyribonucleic acid of Cancer pagurus. II. Template activity for a DNA-dependent DNA polymerase of eukaryotic cells.

The template activity of Cancer pagurus DNA and its two components (poly d(A-T) and main component) in response to a DNA polymerase purified from regenerating rat liver has been studied and compared to the results previously obtained with synthetic templates. In the double-stranded native state, whole crab DNA and the main component were poor templates. Their replication was increased by thermal denaturation and inhibited by actinomycin. Like the synthetic copolymer poly[d(A-T).d(T-A)], native crab poly d(A-T) could be copied and its duplication was not inhibited by actinomycin. The structural difference between native poly d(A-T) Form I, isolated on a density gradient, and partially renatured poly d(A-T) Form II, isolated on hydroxylapatite, resulted in a modification of their template activity. The kinetic studies of [(3)H] dGMP and [(3)H] dAMP incorporation confirmed the importance of single-stranded regions (particulary dC regions) in the initiation of the in vitro duplication.

Differential over-expression of mdr1 genes in multidrug-resistant rat glioblastoma cell lines selected with doxorubicin or vincristine.

We have compared the pharmacological and molecular characteristics of 2 cell lines derived from the C6 rat glioblastoma, and selected for resistance either to doxorubicin (C6 0.5 line) or to vincristine (C6 IV line). Each line displays a preferential 400-fold resistance towards the drug used for selection, the C6 IV line being especially weakly resistant to doxorubicin (13-fold). Verapamil completely restored doxorubicin sensitivity in the C6 IV line as well as vincristine resistance in the C6 0.5 line, but could not completely reverse doxorubicin resistance in the C6 0.5 line or vincristine resistance in the C6 IV line. This suggests that specific mechanisms of resistance against each drug were added to a common P-glycoprotein-mediated multidrug-resistance mechanism. Doxorubicin efflux was total within 2 hr in the C6 IV line, whereas it remained 8 to 10% of drug in the C6 0.5 line 4 hr after drug removal, despite a more rapid efflux of the drug in the first 30 min. This 2-compartment behavior could be related to a special sub-cellular distribution of doxorubicin in C6 0.5 cells. Northern and Western blot analysis of the mdrI gene and of the P-glycoprotein expressed by the 2 resistant cell lines made it possible to quantify their degree of over-expression; when compared with the C6 wild strain, the C6 0.5 line over-expressed both the mdrI gene and the P-glycoprotein to a slightly higher level than the C6 IV line. Northern and Western blot analysis also suggested that C6 0.5 cell preferentially over-expressed the mdrIa gene, whereas the C6 IV cells preferentially over-expressed the mdrIb gene. This differential over-expression was confirmed after polymerase-chain-reaction amplification of the cDNA sequences transcribed from total RNA extracted from the 2 lines. It can be concluded therefore that the mdrIa gene product is more efficient than the mdrIb gene product in extruding anti-cancer drugs from the cells; and that the mdrIb gene product might preferentially extrude vincristine rather than doxorubicin.