Modulation of human P-glycoprotein epitope expression by temperature and/or resistance-modulating agents.

Three monoclonal antibodies (mAb), MRK16, MM4.17 and MC57, directed against distinct epitopes on the external domain of human P-glycoprotein (Pgp), were used to follow its expression on multidrug resistant (MDR)-cells. The linear MM4.17 epitope and conformational MRK16 epitope showed a 4-fold higher expression at 37 degrees C than at 4 degrees C, while the detection of the conformational MC57 epitope did not change. Inhibition of Pgp function, by a short pretreatment of the MDR-cells with resistance-modulating agents (RMA), such as SDZ PSC 833 and SDZ 280-446, could not be related to depletion of Pgp from the cell surface, since their expression of the MM4.17 and MRK16 epitopes was found unchanged. However, a substantially higher expression of MC57 epitopes was found on RMA-treated cells than on untreated ones. Since this effect correlated to the strength of different RMA in reversing the MDR phenotype, MC57 epitopes might be more efficiently expressed on inactivate(d) forms of the Pgp molecules, suggesting that RMA might inhibit Pgp function by disturbing the conformation of individual Pgp molecules, their topographical distribution or polymerization status in the membrane.

SDZ 280-125: a cyclopeptolide endowed with an in vitro cyclosporin A-like profile of activity for the reversion of the P-glycoprotein-mediated multidrug resistance of tumor cells.

Tumor cells whose multidrug resistance is caused by the P-glycoprotein (Pgp) mediated anti-cancer drug (ACD) efflux can be chemosensitized by cyclosporins, whose derivatives were found to display a whole range of resistance-modulating activities. Similarly, derivatives of the non-immunosuppressive natural fungus cyclic peptolide SDZ 90-215 were recently shown to display a broad range of chemosensitizing activities. With highly resistant cells expressing high levels of Pgp, one such compound (SDZ 280-125) was shown here to restore both a normal sensitivity to the growth-inhibitory effects of ACD and a normal retention of an anthracycline antibiotic. With both read-outs, SDZ 280-125 activity was about 3-fold that of cyclosporin A (CsA). SDZ 280-125 also displayed the same profile of chemosensitization as CsA for different ACD classes.

Atypical multi-drug resistance (MDR): low sensitivity of a P-glycoprotein-expressing human T lymphoblastoid MDR cell line to classical P-glycoprotein-directed resistance-modulating agents.

Verapamil, cyclosporin A (CsA), the cyclosporin derivative SDZ PSC 833 and the novel cyclopeptolide SDZ 280-446 were tested for their capacity to chemosensitize a P-glycoprotein (Pgp)-expressing multi-drug resistant (MDR) variant of the CEM human T lymphoblastoid cell subline (CCRF ACTD 400+). That MDR-CEM cell subline had been previously selected for MDR by actinomycin D and displayed a very high resistance phenotype: 3700-fold for actinomycin D, 3900-fold for vincristine, 1200-fold for taxol, 1000-fold for daunomycin (DAU) and 400-fold for colchicine. Interestingly, these MDR-CEM cells displayed little chemosensitization by resistance-modulating agents (RMA) which presumably work by inhibiting Pgp function. These MDR-CEM cells displayed virtually no chemosensitization by 1 microM verapamil or 1 microgram/ml (about 0.8 microM) CsA, whereas their chemosensitization for different anti-cancer drugs (ACD) was rather stable (from 51- to 82-fold) with 1 microgram/ml (about 0.8 microM) SDZ 280-446, while being very unbalanced (from 5- to 38-fold) with 1 microgram/ml (about 0.8 microM) SDZ PSC 833. Exposure of the MDR-CEM cells to Pgp-directed RMAs, during their loading with DAU (DAU-loading phase), hardly restored DAU retention: SDZ 280-446 being as poorly active as SDZ PSC 833, and about only 3- and 4-fold more active than CsA and verapamil. In contrast, SDZ PSC 833 treatment of human MDR-KB and MDR-LoVo cell lines under the same conditions could restore most or all the DAU retention shown by the parental (Par) cells, in spite of their high level of resistance. By keeping the MDR-CEM cells in the presence of RMA throughout the experiment (both DAU-loading and DAU-efflux phases), a better DAU retention could be restored by the different RMAs used, their order of relative restoration activity being SDZ 280-446 3- to 4-fold > SDZ PSC 833 3- to 10-fold > CsA 2- to 4-fold > verapamil. Nevertheless, the level of DAU retention restored in the MDR-CEM cells reached a plateau at 50% of the Par-CEM cell level. Therefore, although the MDR-CEM cells expressed easily detectable membranous Pgp molecules and probably used them for DAU efflux, they displayed an additional efflux mechanism that was not sensitive to the Pgp inhibitors.

Reversion of the P-glycoprotein-mediated multidrug resistance of cancer cells by FK-506 derivatives.

FK-506 is a resistance-modulating agent (RMA) for tumor cells whose multidrug resistance (MDR) involves a P-glycoprotein (Pgp)-mediated anti-cancer drug efflux. The family of FK-506 relatives and derivatives includes analogs which display a whole range of chemosensitizing strengths, from no detectable RMA activity to a complete reversion of the MDR phenotype. Similarly, FK-506 analogs display a whole range of immunosuppressive activities, including inactive ones. FK-506 was compared for RMA activity with 11 FK-506 analogs which were at least 20-fold less active than FK-506 for the inhibition of the bi-directional mixed lymphocyte reaction displayed the whole range of RMA activity. One such strong RMA derivative of FK-506 (SDZ 280-629) was further shown able to restore completely daunomycin retention by highly resistant MDR P388 tumor cells.

Restoration of taxol sensitivity of multidrug-resistant cells by the cyclosporine SDZ PSC 833 and the cyclopeptolide SDZ 280-446.

BACKGROUND: Taxol, a promising agent for the treatment of cancer, has entered phase II clinical trials. Nevertheless, it belongs to the class of compounds that show impaired retention in multidrug-resistant cells expressing P-glycoprotein (Pgp), a drug efflux pump. Chemosensitizers like verapamil modulate multidrug resistance by interfering with the efflux action of Pgp and thus can decrease drug resistance or can restore drug sensitivity by restoring normal drug accumulation and distribution within the multidrug-resistant tumor cell. The two strongest, nearly equipotent chemosensitizers identified to date are the cyclosporine derivative SDZ PSC 833 and the semisynthetic cyclopeptolide SDZ 280-446. PURPOSE: This study was designed to investigate the capacities of verapamil, SDZ PSC 833, and SDZ 280-446 to decrease resistance of two multidrug-resistant cell lines to taxol. METHODS: We studied in vitro the growth of two multidrug-resistant tumor cell lines displaying high resistance to taxol: multidrug-resistant Chinese hamster ovary cells and murine monocytic leukemia P388 cells. We determined the taxol concentration that produced 50% inhibition of cell growth (IC50) in the two multidrug-resistant cell lines and in the parent cell lines, in the presence of a range of chemosensitizer concentrations (0-30 microM). IC50 values were determined in the presence and in the absence of verapamil, SDZ PSC 833, or SDZ 280-446. RESULTS: At nontoxic concentrations (0.3-1 microM), SDZ PSC 833 and SDZ 280-446 produced an almost complete reversal of the high taxol resistance of the multidrug-resistant tumor cells, whereas only partial restoration of sensitivity to taxol was achieved with verapamil. CONCLUSION: SDZ PSC 833 and SDZ 280-446 can restore the normal taxol sensitivity of highly resistant multidrug-resistant tumor cells. IMPLICATIONS: The combination of taxol with SDZ PSC 833 or SDZ 280-446 may be recommended for treatment of multidrug-resistant cancers.

Modulation of adoptively transferred viable motheaten pathology in sublethally irradiated normal recipient mice by normal hematopoietic cells.

"Adoptive mev" chimeras were created by grafting hematopoietic cells (HC) from B6 viable motheaten (mev) mice into bg or wild sublethally irradiated (SI) mice: the chimeras developed mev-type symptoms such as paw inflammation and necrosis, lung damage, thymus atrophy, and high serological IgM concentration and autoantibody levels as well as rapid death. The phenotype of adoptive mev mice could be obtained even after two to three successive passages into SI mice (Kuntz et al., 1991. Immunology 73, 356). In the present study, mixed HC transfer experiments showed that bg or wild HC could not prevent or delay neither the serological symptoms nor the pathology conferred by cotransferred mev HC. Nevertheless, when cotransferred with adoptive mev chimera HC, bg or wild HC could block the development of the pathology and the morbidity, although only delayed the emergence of the serological abnormalities. This shows that the differentiation of mev HC in a bg or wild normal-type environment does not allow the maintenance of all mev HC-dependent abnormalities.

Serum immunoglobulin isotype profile of viable and non viable lymphoid cell chimaeras made with nude athymic lpr (lymphoproliferation) mouse recipients.

C57BL/6 mice (B6) which are homozygous at the nu (nude, athymic) and lpr (lymphoproliferation) locus (B6 nulpr) are short-lived. We showed previously that increased survival could be obtained by grafting lymphoid cells from euthymic lpr-homozygous B6 mice (B6 lpr) mice ([lpr----nulpr] chimaeras), but curiously enough not from normal (B6 wild) mice ([wild----nulpr] chimaeras). Moreover female, but not male, [lpr----nulpr] chimaeras developed spleen and lymph node enlargement. In the present paper the distribution and absolute concentrations of all serum immunoglobulin (Ig) isotypes have been determined in these chimaeras and their controls. All chimaeras displayed whole serum Ig levels higher than those of B6 wild mice, suggesting a successful reconstitution of the athymic recipients by the grafted lymphoid cells, but two types of chimaeras were peculiar. The short-lived [wild----nulpr] chimaeras showed a proportion of IgM as high as ungrafted B6 nulpr mice, suggesting a deficient down-regulation of IgM production by the grafted B6 wild-type lymphoid cells. The [lpr----nulpr] female chimaeras recovered a long lasting overexpression of all Ig isotypes, like B6 lpr mice, while all the other chimaeras showed a transient overexpression only. Since neither lymphadenopathy nor persistent increase of serum Ig levels were observed in [lpr----nu] chimaeras, our data confirmed the need for a genetically lpr host to allow the significant development of the lpr syndrome.

SDZ 280-446, a novel semi-synthetic cyclopeptolide: in vitro and in vivo circumvention of the P-glycoprotein-mediated tumour cell multidrug resistance.

SDZ 280-446 is a semi-synthetic derivative of a natural cyclic peptolide. Its ability to sensitise in vitro tumour cells whose resistance is due to P-glycoprotein-mediated anticancer-drug efflux was shown using four different pairs of parental drug-sensitive (Par-) and multidrug-resistant (MDR-) cell lines, from three different species (mouse, human, Chinese hamster) representing four different cell lineages (monocytic leukaemia, nasopharyngeal epithelial carcinoma, colon epithelial carcinoma, ovary fibroblastoid carcinoma), and using four different drug classes (colchicine, vincristine, daunomycin/doxorubicin and etoposide). By measuring its capacity to restore normal drug sensitivity of MDR-cells in culture in vitro, it appeared that SDZ 280-446 belongs to the same class of very potent chemosensitisers as the cyclosporin derivative SDZ PSC 833: both are about one order of magnitude more active than cyclosporin A (CsA), which is itself about one order of magnitude more active than other known chemosensitisers (including verapamil, quinidine and amiodarone which have already entered clinical trials in MDR reversal). Low concentrations of SDZ 280-446 could also restore cellular daunomycin retention in MDR-P388 cells to the levels found in the Par-P388 cells. SDZ 280-446 was also effective as a chemosensitiser when given orally in vivo. In a syngeneic mouse model, combined therapy with vinca alkaloids given i.p. and SDZ 280-446 given per os for 5 consecutive days significantly prolonged the survival of MDR-P388 tumour-bearing mice, when compared with mice receiving vinca alkaloids alone. Another protocol, using three cycles of i.p. doxorubicin at 4 day intervals, could also not increase MDR-P388 tumour-bearing mouse survival unless the mice received SDZ 280-446 orally 4 h before each doxorubicin injection. Though only very few combined therapy treatment protocols have been tested so far, clear increases in survival time of MDR-tumour-bearing mice were regularly obtained, leaving hope for major improvement of the therapy using other dosing schedules.

In vivo circumvention of P-glycoprotein-mediated multidrug resistance of tumor cells with SDZ PSC 833.

The new nonimmunosuppressive cyclosporin analogue, SDZ PSC 833, is a very potent multidrug-resistance modifier. In vitro, it was shown to be at least 10-fold more active than cyclosporin A (Sandimmune), itself more active than verapamil, on most P-glycoprotein-expressing multidrug-resistant (MDR) tumor cell lines. In vivo, SDZ PSC 833 was tested in a few protocols of combined therapy with either Vinca alkaloids or doxorubicin as anticancer drugs, using the homologous tumor-host system (P388 cells of DBA/2 origin grafted into DBA/2 or B6D2F1 mice). Although these MDR-P388 tumor cells belong to a highly resistant variant that in vitro required about 150-fold more anticancer drug for 50% cell growth inhibition than the parental P388 cells, significant prolongation of survival times of the MDR-P388 tumor-bearing mice was obtained when treated with a combination of SDZ PSC 833 p.o. were otherwise ineffective doses of anticancer drugs given i.p. This chemosensitizing effect of SDZ PSC 833 was dose-dependent and was most effective in a protocol combining administration of SDZ PSC 833 p.o. 4 h before a doxorubicin i.p. injection: in comparison with the survival of MDR-P388 tumor-bearing mice treated with the anticancer drug alone, the pretreatment with SDZ PSC 833 at 25 and 50 mg/kg gave 2- to 3-fold increases of survival times. Since the MDR-P388 tumor cells used in our studies belong to a highly resistant variant, with a much higher degree of drug resistance than the one known to occur in cancer patients, SDZ PSC 833 appears to be a very promising chemosensitizer.

Adoptive transfer of viable motheaten pathology in sublethally irradiated beige recipient mice.

B6 viable motheaten (mev) mice are short-living autoimmune and immunodeficient mice. Most of the mev pathology is transferable into the B6 beige (bg) irradiated mouse by injection of mev haematopoietic cells. Indeed there is a rapid establishment of lung damage, thymus aplasia, inflammation and necrosis of peripheral joints, an increase of serological IgM concentrations and autoantibody levels as well as a rapid death. This mev-type pathology can be again obtained by transferring haematopoietic cells of these [mev----bg] chimeras into other irradiated bg mice, although the appearance of these serological and phenotypical symptoms seems to be more and more delayed when doing cascade transfers.

Immunoglobulin isotypes of C57BL/6 nu/nu, lpr/lpr mice. Lack of direct intrinsic effect of the lpr gene on B cell hyperactivity.

The absolute concentrations of mouse immunoglobulin (Ig) heavy chain isotypes were determined by specific ELISAs in the serum of C57BL/6 (B6) mice doubly homozygous at the nude (nu) and the lymphoproliferation (lpr) locus (B6 nu, lpr mice), and compared with normal B6 nu mice. The distribution and the absolute concentrations of all Ig isotypes were found to be very similar in B6 nu, lpr and B6 nu mice, for both sexes and with similar increases in titers with ageing. Thus, the major part of the severe autoimmunity and hyperglobulinemia characteristic of the lpr syndrome of euthymic B6 lpr mice, including their elevated titers of thymus-independent IgM and IgG3 isotypes, is abrogated by the nude mutation, an effect of which is the lack of thymus differentiation. Though a postulated intrinsic activity of the lpr gene directly on B cell hyperactivity cannot be discarded, its expression would then require the presence of either the thymus or of T cells or of other cells or factors whose expression is also abrogated by the homozygosity at the nude locus.

Lymphoid cell transfers between adult C57BL/6 mice differing at the lpr and/or nu locus. Humoral immunity phenotype of the chimeras.

Congenic C57BL/6 mice (B6) homozygous only at the nu (nude, athymic) locus (B6 nu) or also at the lpr (lympho-proliferation) locus (B6 nu, lpr) were used as recipients for the transfer of spleen and/or lymph node cells from either normal B6 mice (B6+) or lpr-homozygous B6 mice (B6 lpr). Highly increased survival was obtained for [B6 lpr----B6 nu, lpr] chimeras, but not for [B6+----B6 nu, lpr] and [B6 nu----B6 nu, lpr] chimeras. All long-term survivors that were killed showed an increased responsiveness to the T-cell mitogen concanavalin A (Con A) but no change of responsiveness to the B-cell mitogen lipopolysaccharide (LPS). Some enlargement of spleen and lymph nodes was observed only in some specific [B6----B6 nu, lpr] chimeras (female recipients of spleen cells). Within a few weeks after cell grafting, all but [B6 nu----B6 nu, lpr] chimeras developed highly increased levels of serum immunoglobulins, as well as a higher occurrence of anti-single-stranded (ss) DNA containing sera. These chimeras had been constructed in order to dissect the components of the lpr phenotype etiopathology (primordial involvement of B and/or T lineage cells, lymphoid and/or environmental influences). Though the evolution of serological parameters showed some chimera type-specific features, it seems difficult to reconstitute the entire expression of the lpr phenotype.

Partial expression of the lpr locus in the heterozygous state: presence of autoantibodies.

B6 mice heterozygous at the lpr locus (B6 +/lpr and B6 lpr/+) were compared with lpr homozygous mice (B6 lpr/lpr) and control mice (B6 +/+) for levels of serum immunoglobulin (Ig), presence of autoantibodies and rate of B-cell membrane immunoglobulin (mIg) capping. The total serum Ig levels in B6 +/lpr and B6 lpr/+ mice remained much below the high titres found in B6 lpr/lpr mice, and were close to the titres found in B6 +/+ mice. However, the presence of anti-single-stranded (ss) DNA antibodies and of anti-nuclear antibodies (ANA) was detected in most B6 +/lpr and B6 lpr/+ mice, although less frequently and in lower titres than in B6 lpr/lpr mice. The rate of mIg capping was higher in B6 +/lpr and B6 lpr/+ mice than in B6 +/+ mice, but the acceleration of the capping process remained inferior to the one found in B6 lpr/lpr mice. Therefore, the lpr locus is not totally recessive: some B-cell hyperactivity is expressed in the heterozygous state. This is in contrast with its lack of expression at the level of lympho-proliferation of the lpr-characteristic T-cell subset: none of the lpr heterozygous B6 mice displayed detectable lymphadenopathy.

Radiation therapy of spontaneous autoimmunity: a review of mouse models.

The classical types of generalized autoimmune disease in man are systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Several murine strains which develop SLE and sometimes RA-like diseases are now available. They should help in the understanding of the etiopathology of SLE and RA. Basically two main therapeutic strategies which use solely irradiation have been tried; one being sublethal whole-body irradiation (WBI) and the other fractionated total lymphoid irradiation (TLI). Other protocols which combine lethal WBI and stem cell transplantation have often been attempted. It was regularly found that the bone marrow transplant (BMT) dictates the immune status of the recipient. This paper reviews the data published about NZB, NZB/W, BXSB and MRL mice in this context.

Lymphoid cell transfers between adult C57BL/6 mice differing at the LPR locus. Lack of lymphadenopathy transfer and effects on host survival.

"1 pr" is an autosomal recessive locus which determines the lymphoproliferation of an abnormal T cell subset ("T lpr" cell subset). Though a thymus is necessary for the initiation of the lymphadenopathy, adult thymectomy does not interfere with the development of disease. C57BL/6 (B6) mice (either treated with cyclophosphamide or not), lpr heterozygous at the lpr locus, or not), and nu homozygous B6 mice (either homozygous at the lpr locus, or not) are refractory to the growth and massive proliferation of grafted cells of the aberrant T lpr cell subset, which polyclonally expands in lpr homozygous B6 mice. Their lack of expansion in B6 nu, lpr mice is surprising, since such animals may develop the lymphadenopathy under certain circumstances (thymus grafting). While the injection of normal B6 lymphoid cells does not improve the health of the B6 nu, lpr mice, but may even accelerate their wasting, the injection of B6 lpr lymphoid cells into B6 nu, lpr mice causes, after a transient wasting, a remarkable prolongation of survival. B6 nu recipients of B6 lpr lymphoid cells show no sign of wasting and survive like recipients of normal B6 (B6+) cells. Thus the "lpr type" lymphoproliferative potential is neither simply carried by the T lpr subset cells themselves, nor simply determined by the lpr environment of athymic, lpr homozygous mice, and it is also not readily reconstituted by grafting T lpr cells in athymic lpr mice.