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We previously showed that adhesion of myeloma cells to fibronectin (FN) by means of beta1 integrins causes resistance to certain cytotoxic drugs. The study described here found that adhesion of U937 human histiocytic lymphoma cells to FN provides a survival advantage with respect to damage induced by the topoisomerase (topo) II inhibitors mitoxantrone, doxorubicin, and etoposide. Apoptosis induced by a topo II inhibitor is thought to be initiated by DNA damage. The neutral comet assay was used to determine whether initial drug-induced DNA damage correlated with cellular-adhesion-mediated drug resistance. Cellular adhesion by means of beta1 integrins resulted in a 40% to 60% reduction in mitoxantrone- and etoposide-induced DNA double-strand breaks. When the mechanisms regulating the initial drug-induced DNA damage were examined, a beta1 integrin-mediated reduction in drug-induced DNA double-strand breaks was found to correlate with reduced topo II activity and decreased salt-extractable nuclear topo IIbeta protein levels. Confocal studies showed changes in the nuclear localization of topo IIbeta; however, alterations in the nuclear-to-cytoplasmic ratio of topo IIbeta in FN-adhered cells were not significantly different. Furthermore, after a high level of salt extraction of nuclear proteins, higher levels of topo IIbeta-associated DNA binding were observed in FN-adhered cells than in cells in suspension. Together, these data suggest that topo IIbeta is more tightly bound to the nucleus of FN-adhered cells. Thus, FN adhesion by means of beta1 integrins appears to protect U937 cells from initial drug-induced DNA damage by reducing topo II activity secondarily to alterations in the nuclear distribution of topo IIbeta.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by slow-growing plasma cells in the bone marrow (BM). Patients with MM typically respond to initial chemotherapies; however, essentially all progress to a chemoresistant state. Factors that contribute to the chemorefractory phenotype include modulation of free radical scavenging, increased expression of drug efflux pumps, and changes in gene expression that allow escape from apoptotic signaling. Recent data indicate that arsenic trioxide (As(2)O(3)) induces remission of refractory acute promyelocytic leukemia and apoptosis of cell lines overexpressing Bcl-2 family members; therefore, it was hypothesized that chemorefractory MM cells would be sensitive to As(2)O(3). As(2)O(3) induced apoptosis in 4 human MM cell lines: 8226/S, 8226/Dox40, U266, and U266/Bcl-x(L). The addition of interleukin-6 had no effect on cell death. Glutathione (GSH) has been implicated as an inhibitor of As(2)O(3)-induced cell death either through conjugating As(2)O(3) or by sequestering reactive oxygen induced by As(2)O(3). Consistent with this possibility, increasing GSH levels with N-acetylcysteine attenuated As(2)O(3) cytotoxicity. Decreases in GSH have been associated with ascorbic acid (AA) metabolism. Clinically relevant doses of AA decreased GSH levels and potentiated As(2)O(3)-mediated cell death of all 4 MM cell lines. Similar results were obtained in freshly isolated human MM cells. In contrast, normal BM cells displayed little sensitivity to As(2)O(3) alone or in combination with AA. Together, these data suggest that As(2)O(3) and AA may be effective antineoplastic agents in refractory MM and that AA might be a useful adjuvant in GSH-sensitive therapies. (Blood. 2001;98:805-813)

We have previously shown that selection for resistance to the anthracenes, doxorubicin or mitoxantrone, results in coselection for resistance to CD95-mediated apoptosis (Landowski et al: Blood 89:1854, 1997). In the present study, we were interested in determining if the converse is also true; that is, does selection for CD95 resistance coselect for resistance to chemotherapeutic drugs. To address this question, we used two isogenic models of CD95-resistant versus CD95-sensitive cell lines: 8226/S myeloma cells selected for resistance to CD95-mediated apoptosis; and K562 cells expressing ectopic CD95. Repeated exposure of the CD95-sensitive human myeloma cell line, 8226/S, to agonistic anti-CD95 antibody resulted in a cell line devoid of CD95 receptor surface expression and completely resistant to CD95-mediated apoptosis. Multiple clonal populations derived from the CD95-resistant cell line showed no difference in sensitivity to doxorubicin, mitoxantrone, Ara-C, or etoposide, demonstrating that cross-resistance between Fas-mediated apoptosis and drug-induced apoptosis occurs only when cytotoxic drugs are used as the selecting agent. Using the inverse approach, we transfected the CD95-negative cell line, K562, with a CD95 expression vector. Clones expressing variable levels of cell-surface CD95 were isolated by limiting dilution, and analyzed for sensitivity to CD95-mediated apoptosis and response to chemotherapeutic drugs. We show that CD95 surface expression confers sensitivity to CD95-mediated apoptosis; however, it does not alter response to chemotherapeutic drugs. Similarly, doxorubicin-induced activation of caspases 3 and 8 was identical in the CD95-sensitive and CD95-resistant cell lines in both isogenic cell systems. In addition, prior treatment with the CD95 receptor-blocking antibody, ZB4, inhibited CD95-activated apoptosis in 8226/S cells, but had no effect on doxorubicin cytotoxicity. These results show that CD95 and chemotherapeutic drugs use common apoptotic effectors, but the point of convergence in these two pathways is downstream of CD95 receptor/ligand interaction.

Tumor cells that survive initial courses of chemotherapy may do so by acquiring a multidrug-resistant phenotype. This particular mechanism of drug resistance may also confer resistance to physiological effectors of apoptosis that could potentially reduce the efficacy of immune therapies that use these pathways of cell death. We have previously demonstrated high efficacy for a cytokine-based tumor cell vaccine in a murine MPC11 myeloma model. In the present study, the effects of this vaccination were compared in MPC11 cells and their isogenic sublines selected for mdr1/P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). Immunization with MPC11 cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 (IL-12) led to long-lasting protection of mice against subcutaneous (sc) challenge with both parental cells or their MDR variants. Similarly, immunization with GM-CSF/IL-12-transfected MDR sublines caused rejection of transplantation of both parental cells and the MDR sublines. Whereas MPC11 cells and their MDR variants were resistant to APO-1/CD95/Fas ligand, the immunization generated potent granzyme B/perforin-secreting cytotoxic T lymphocytes (CTLs) that were similarly effective against both parental and isogenic MDR cells. We conclude that MDR mediated by mdr1/Pgp did not interfere with lysis by pore-forming CTLs. Immunotherapy based on pore-forming CTLs may be an attractive approach to the treatment of drug-resistant myeloma.

Integrin-mediated adhesion influences cell survival and may prevent programmed cell death. Little is known about how drug-sensitive tumor cell lines survive initial exposures to cytotoxic drugs and eventually select for drug-resistant populations. Factors that allow for cell survival following acute cytotoxic drug exposure may differ from drug resistance mechanisms selected for by chronic drug exposure. We show here that drug-sensitive 8226 human myeloma cells, demonstrated to express both VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) integrin fibronectin (FN) receptors, are relatively resistant to the apoptotic effects of doxorubicin and melphalan when pre-adhered to FN and compared with cells grown in suspension. This cell adhesion mediated drug resistance, or CAM-DR, was not due to reduced drug accumulation or upregulation of anti-apoptotic Bcl-2 family members. As determined by flow cytometry, myeloma cell lines selected for drug resistance, with either doxorubicin or melphalan, overexpress VLA-4. Functional assays revealed a significant increase in alpha4-mediated cell adhesion in both drug-resistant variants compared with the drug-sensitive parent line. When removed from selection pressure, drug-resistant cell lines reverted to a drug sensitive and alpha4-low phenotype. Whether VLA-4-mediated FN adhesion offers a survival advantage over VLA-5-mediated adhesion remains to be determined. In conclusion, we have demonstrated that FN-mediated adhesion confers a survival advantage for myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis. This finding may explain how some cells survive initial drug exposure and eventually express classical mechanisms of drug resistance such as MDR1 overexpression.

CD95 (Fas)-induced apoptosis plays a critical role in the elimination of activated lymphocytes and induction of peripheral tolerance. Defects in CD95/CD95L (Fas-Ligand)-apoptotic pathway have been recognized in autoimmune lymphoproliferative diseases (ALPS) and lpr or gld mice and attributed to CD95 and CD95L gene mutations, respectively. Large granular lymphocyte (LGL) leukemia is a chronic disease characterized by a proliferation of antigen-activated cytotoxic T lymphocytes. Autoimmune features such as hypergammaglobulinemia, rheumatoid factor, and circulating immune complexes are common features in LGL leukemia and ALPS. Therefore, we hypothesize that expansion of leukemic LGL may be secondary to a defective CD95 apoptotic pathway. In this study, we investigated expression of CD95 and CD95L in 11 patients with CD3(+) LGL leukemia and explored the apoptotic response to agonistic CD95 monoclonal antibody (MoAb). We found that leukemic LGL from each patient expressed constitutively high levels of CD95/CD95L, similar to those seen in normal activated T cells. However, cells from 9 of these 11 patients were totally resistant to anti-CD95-induced apoptosis. Similarly, cells were resistant to anti-CD3-MoAb-triggered cell death. Lack of anti-CD95-induced apoptosis was not due to mutations in the CD95 antigen. Leukemic LGL were not intrinsically resistant to CD95-dependent death, because LGL from all but 1 patient underwent apoptosis after phytohemagglutinin/interleukin-2 activation. The patient whose leukemic LGL were intrinsically resistant to CD95 had an aggressive form of LGL leukemia that was resistant to combination chemotherapy. These findings that leukemic LGL are resistant to CD95-dependent apoptosis despite expressing high levels of CD95 are similar to observations made in CD95L transgenic mice. These data suggest that LGL leukemia may be a useful model of dysregulated apoptosis causing human malignancy and autoimmune disease.

Programmed cell death, or apoptosis, is well documented as a physiological means of eliminating activated lymphocytes and maintaining immune homeostasis. Apoptosis has also been implicated in the targeting of tumor cells by cytotoxic T lymphocytes and natural killer cells. One of the two primary mechanisms used in cell-mediated cytotoxicity is the Fas/FasLigand system. Activated or transformed cells expressing the Fas antigen on their surface are susceptible to killing by immune effector cells that express the Fas ligand. Many neoplastic cells, including those derived from patients with multiple myeloma, express Fas antigen on their surface, but do not undergo apoptosis in response to antigen crosslinking. One possibility for the lack of Fas-mediated apoptosis includes mutations in the Fas antigen. Loss of function mutations in the Fas antigen have been associated with congenital autoimmune disease in humans, and have been defined as the genetic defect the in lpr mice. Mutations in the Fas antigen have not been previously described in cancer patients. In this study, we show that mutations occur in the Fas antigen which may cause loss of function and contribute to the pathogenesis of the neoplastic disease, multiple myeloma. Using reverse transcriptase-polymerase chain reaction (RT-PCR), single-stranded conformation polymorphism (SSCP) analysis, and DNA sequencing, we examined the cDNA structure of the Fas antigen in 54 bone marrow (BM) specimens obtained from myeloma patients. Six patient specimens (11%) did not express detectable levels of Fas antigen mRNA. Of the 48 BM specimens which did express Fas antigen, 5 (10%) displayed point mutations. All of the mutations identified were located in the cytoplasmic region of the Fas antigen known to be involved in transduction of an apoptotic signal. Two separate individuals demonstrated an identical mutation at a site previously shown to be mutated in the congenital autoimmune syndrome, ALPS. One patient exhibited a point mutation at a site only two amino acids removed from the documented lesion of the lprcg mouse. Although the functional status of these point mutations remains to be determined, we propose that Fas antigen mutations may contribute to the pathogenesis and progression of myeloma in some patients.

Decreased topoisomerase II (Topo II) activity results in resistance to antineoplastic agents targeting this enzyme. Dox1V derived from human multiple myeloma RPMI 8226 demonstrated a 4-fold resistance to doxorubicin in the absence of MDR1 overexpression or topo II mutations (Futscher B.W., Foley N., Gleason-Guzman M., Meltzer P.S., Sullivan D.M., and Dalton W.S., Int'l. J. Cancer, 66: 520-5, 1996.). Consistent with its drug resistant phenotype, a 2- to 3-fold decrease in topo II expression was identified. To investigate the molecular basis for decreased topo II expression in Dox1V, a semi-quantitative analysis of Topo II activity, protein level and mRNA transcript were performed. The results demonstrated that reduced Topo II activity is due to a decreased mRNA level. Southern blot and sequencing experiments revealed wild-type sequence of the topo II promoter in the drug resistant cells. Transient gene expression assays demonstrated that topo II is transcriptionally down-regulated in Dox1V independent of the promoter sequence of the endogenous alleles. Instead, the activity of a ubiquitous transcription factor CP-1 (NF-Y) interacting with the topo II promoter is decreased. The decrease in CP-1/NF-Y activity in Dox1V is correlated well with the decrease in topo II transcriptional activity, transcript level, Topo II protein and enzyme activity. Therefore, transcriptional down-regulation resulted from a reduced CP-1/NF-Y activity is responsible for decreased topo II expression in Dox1V cells.

Recent evidence has supported the hypothesis that chemotherapeutic drugs and radiation induce an apoptotic pathway that requires the active participation of the cell. One pathway of apoptosis in malignant lymphoid cells is mediated by the Fas antigen. We studied the human myeloma (8226) and T-cell leukemia (CEM) cell lines selected for resistance to the anthracenes, doxorubicin or mitoxantrone, by continuous culture in the presence of either agent. We found that these drug-resistant cell lines were also resistant to Fas-mediated apoptosis in a dose-dependent manner. The degree of resistance to Fas-mediated apoptosis correlated directly with the level of resistance to chemotherapeutic drugs. These observations indicate that, as cancer cell lines develop mechanisms of drug resistance, they may also develop mechanisms of resistance to physiologic signals of apoptosis. Two mechanisms of resistance to Fas-mediated apoptosis were observed in these cell lines. One mechanism was associated with a dose-dependent reduction in the surface expression of Fas antigen. Analysis of RNA by reverse transcriptase-polymerase chain reaction assays showed that the reduction of Fas antigen expression occurred at the level of transcription. A second mechanism of drug resistance showed no decrease of Fas antigen expression; however, the apoptotic response was diminished. In this situation, removal of the chemotherapeutic agent resulted in a partial reversion to chemosensitivity and re-expression of the Fas antigen, but these cell lines did not regain the ability to undergo apoptosis in response to cross-linking by anti-Fas antibody. These findings support the hypothesis that apoptosis mediated by both chemotherapeutic agents and physiologic stimuli may share a common downstream effector. The demonstration that selection for drug resistance in hematopoietic cell lines results in a simultaneous resistance to Fas-mediated apoptosis may have clinical implications in the development of strategies for the treatment of resistant disease. Further analysis of the molecular mechanisms of Fas expression and function will facilitate the design of biological response modifying agents for the treatment of malignancy.

The monoclonal antibody LRP56 recognizes a 110-kD major vault protein (lung-resistance protein [LRP]) overexpressed in several P-glycoprotein-negative (Pgp-), multidrug resistant tumor cell lines. To determine the frequency of LRP overexpression, its prognostic significance, and its relation to Pgp, we analyzed bone marrow specimens from 87 consecutive patients with acute leukemia. Diagnoses included de novo acute myeloid leukemia (AML; 21 patients), leukemia arising from an antecedent hematologic disorder or prior cytotoxic therapy (secondary AML; 27 patients), AML in relapse (29 patients), and blast phase of chronic myeloid leukemia (CML-BP; 10 patients). A granular cytoplasmic staining pattern was detected by immunocytochemistry in 32 (37%) cases, including 7 (33%) de novo AML, 13 (48%) secondary AML, 11 (38%) relapsed AML, and 1 of 10 CML-BP. Among 66 evaluable patients with AML, LRP overexpression was associated with an inferior response to induction chemotherapy (P = .0017). Remissions were achieved in 35% of LRP+ patients as compared with 68% of LRP- patients. Although Pgp adversely affected response in univariate analysis (P = .0414), only LRP had independent prognostic significance when compared in a logistic regression model (P = .0046). Differences in remission duration (P = .075) and overall survival (P = .058) approached significance only for LRP. Sequential specimens from remitting patients receiving treatment with the Pgp modulator cyclosporin-A showed emergence of the LRP phenotype despite a decrease or loss of Pgp at the time of treatment failure (P =.0304). Significant associations were observed between LRP and age greater than 55 years (P = .017), Pgp (P = .040), and prior treatment with mitoxantrone (P = .020) but not with CD34. These findings indicate that overexpression of the novel transporter protein LRP is an important predictor of treatment outcome in AML.

In contrast to its clearly defined role as a multidrug efflux pump in neoplastic cells, the physiologic function of P-glycoprotein (P-gly) in normal cells is unclear. Recent reports identifying P-gly in normal blood and bone marrow suggest that hematopoietic development or function may be dependent on P-gly. To understand the normal function of P-gly in the blood, its level of expression and function must first be quantitated relative to a known standard. In this study, P-gly, MDR1 gene expression, and P-gly function were quantitated in normal leukocytes. P-gly and MDR1 expression were analyzed in individual leukocyte lineages (T-helper, T-suppressor, monocyte, granulocyte, B-lymphocyte, NK cell) from normal volunteers. P-gly on the cell surface was detected by fluorescent double-labeling for lineage (CD4, CD8, CD14, CD15, CD19, CD56, respectively) and P-gly (MRK16) with analysis by flow cytometry and in some cases immunoblot analysis. MDR1 mRNA analysis on purified lineages was performed using quantitative reverse transcription-polymerase chain reaction. P-gly function was determined for each lineage using dual-labeling for lineage and P-gly substrate (rhodamine 123). The P-gly expressing human myeloma cell line, 8226/Dox6, was used as a reference of comparison for levels of P-gly, MDR1 mRNA, and function. CD56+ cells expressed the highest levels of MDR1 mRNA followed by CD8+ > CD4+ approximately equal to CD15+ > CD19+ > CD14+, with percentage values relative to Dox6 of 49%, 17%, 8%, 8%, 4%, and 2%, respectively. The assays for P-gly immunofluorescence and function correlated well with mRNA analysis except for CD15+ cells (granulocytes), which showed a moderate MDR1 mRNA level with a lack of both function and surface P-gly staining. Granulocyte membranes did show P-gly on immunoblot analysis when probed with either C219 or JSB1. We conclude that (1) P-gly and the MDR1 mRNA are expressed in normal leukocytes, (2) this P-gly expression is lineage specific with relatively high levels among CD56+ cells, and (3) the expression of P-gly in granulocytes is not associated with transport of the P-gly substrate, rhodamine 123, out of the cell.

We have designed a new polymerase chain reaction (PCR) protocol for the quantitation of mdr1 mRNA in cell lines and clinical specimens. This protocol uses an in vitro-generated RNA molecule as an internal standard. This synthetic RNA contains the same mdr1 primer sequences as the cellular mRNA, but yields a different-sized PCR product after amplification. Since a single primer set is used in quantitation, differences in primer efficiency are not a concern. We have used this assay to measure mdr1 expression in a multiple myeloma cell line, 8226/S, its drug resistant variants 8226/dox6 and 8226/dox40, and tumor samples from 10 patients with B-cell malignancies (9 multiple myeloma, 1 chronic lymphocytic leukemia). 8226/S does not express mdr1 mRNA. 8226/dox6 is 10-fold resistant to doxorubicin, and expresses 32 mdr1 mRNA/10 pg cellular RNA. 8226/dox40 is 140-fold resistant to doxorubicin, and expresses 890 mdr1 mRNA/10 pg cellular RNA. Seven of the 10 patients had levels of mdr1 mRNA expression below that seen in the multidrug-resistant, human multiple myeloma cell line, 8226/dox6. Three patients had levels of mdr1 expression comparable to those seen in 8226/dox6. No patient had levels of mdr1 expression close to that seen in 8226/dox40. Sample RNA integrity is assured by PCR analysis of a different, ubiquitous, cell cycle independent, histone variant, H3.3. This assay will be useful for studying low level mdr1 expression in cell lines and clinical specimens.

We have established a reproducible in vivo model of human multiple myeloma in the severe combined immunodeficient (SCID) mouse using both the RPMI 8226 human myeloma cell line and the P-glycoprotein-expressing multidrug-resistant 8226/C1N subline. SCID mice 5 to 8 weeks of age were injected intraperitoneally with either 8226 drug-sensitive or P-glycoprotein-expressing multidrug-resistant myeloma cells (8226/C1N). Tumors were detected within 5 days after injection by the presence of human lambda light chain excretion in the mouse urine. Growth of the tumor was observed primarily in the abdominal cavity with spread to the abdominal organs. The anti-neoplastic agent doxorubicin was effective in treating the drug-sensitive 8226 human-SCID xenografts but had no effect on the multi-drug-resistant 8226/C1N human-SCID xenografts. In the 8226-sensitive xenografts, treatment with doxorubicin resulted in a sharp decline in the concentration of human lambda light chain being excreted in the mouse urine. This correlated with an increased survival of the drug-treated animals. This mouse model offers an in vivo means of evaluating efficacy and toxicity of new therapeutic approaches, including development of chemosensitizers directed against P-glycoprotein in multidrug-resistant myelomas.

Multidrug-resistant (MDR) myeloma patients failing chemotherapy may express P-glycoprotein (PGP), which serves as an efflux pump protecting the neoplastic cells. Unknown is whether PGP expression might relate to prior cytotoxic drug exposure. To address this question, we studied 106 consecutive bone marrow samples from 104 myeloma patients with samples studied either before or after therapy and at the time of relapse. We performed an established immunocytochemical assay of PGP using an MDR-1-specific monoclonal antibody and correlated PGP status with prior chemotherapy dosage. Myeloma patients with no prior therapy had a low incidence of PGP expression (6%, 3/47), whereas those receiving chemotherapy had a significantly higher incidence (43%, 21/49) (P < .0001). A substantially higher incidence of PGP expression (50%, 83%, respectively) occurred when the total vincristine dose exceeded 20 mg and when doxorubicin exceeded 340 mg. In the 11 patients who received both high vincristine and doxorubicin dosages (> 20 mg, > 340 mg total dose) there was 100% incidence of PGP expression in the tumor cells. These data provided the basis for a predictive mathematical model from which dose-related PGP expression normograms were generated. Time with myeloma for PGP-negative patients (mean 33 months) had overlapping confidence limits with PGP-positive patients (mean 42 months), suggesting that disease duration was not a significant variable. PGP expression did not correlate with other clinical factors or immunophenotypic factors. Our findings indicate a strong correlation between PGP expression in myeloma and past chemotherapy in myeloma, in particular, related to prior exposure to the natural product agents vincristine and doxorubicin. Additionally, the proportion of PGP-positive plasma cells was significantly higher in the doxorubicin-treated patients than the nondoxorubicin-treated patients (87.7% v 65.17%; P = .013). Combined high vincristine and doxorubicin total dosage appear highly predictive of PGP expression.

Verapamil was evaluated as a chemosensitizer for reversing multidrug resistance in multiple myeloma both in vitro and in clinical trials. Bone marrows from 59 myeloma patients in relapse were evaluated for several resistance parameters: expression of p-glycoprotein (MDR1), doxorubicin (Adriamycin) and vincristine sensitivity, and the ability of added verapamil to reduce resistance to the cytotoxic agents. We found that verapamil was capable of sensitizing myeloma cells that exhibited resistance to doxorubicin and vincristine in vitro, but did not enhance sensitivity of cells that were drug sensitive (P less than .001). Myeloma cells expressing MDR1 immunohistochemically tended to be more doxorubicin resistant in vitro than MDR1-negative cells. In the clinical trials, 22 patients with myeloma refractory to vincristine-Adriamycin-dexamethasone (VAD) were treated with VAD plus high-dose intravenous verapamil (Ve). Among the 22 patients treated with VAD/Ve, five achieved a partial remission (23%). The median relapse-free survival for the VAD/Ve responders was 5.4 months and their overall survival from the start of VAD/Ve was better than that of the nonresponders. Among the subset of 10 patients whose myeloma cells were MDR1 positive, four responded clinically (40%), whereas none of five patients with MDR1-negative myeloma cells achieved remission with VAD/Ve. We also observed that myeloma cells from three of four VAD/Ve clinical responders exhibited in vitro chemosensitization with verapamil, whereas in vitro verapamil chemosensitization was seen in only one of six clinical nonresponders. Our observations demonstrate that clinical reversal of multidrug resistance can be achieved in some patients with VAD-refractory myeloma with the use of verapamil. In addition to their value in drug development, in vitro tests of MDR1 expression and of chemosensitizers plus cytotoxic drugs on the patients' bone marrow myeloma cells may identify patients who will respond clinically to chemosensitizer-containing regimens. We anticipate that chemosensitizer regimens capable of inhibiting multidrug resistance will play an increasing role in the treatment of hematologic malignancies, including B-cell neoplasms such as multiple myeloma and the non-Hodgkin's lymphomas.

In an effort to develop a clinically useful approach to overcoming P-glycoprotein-mediated multidrug resistance (MDR1), we evaluated combined chemosensitization with verapamil and quinine in a multidrug-resistant (MDR) human myeloma cell line model. In clonogenic assay, verapamil was used at concentrations from 0.1 to 1.0 micrograms/mL, bracketing the plasma levels achieved by oral administration and high-dose intravenous (IV) infusion, respectively. The dose of quinine was held constant at 1.0 micrograms/mL, a plasma concentration readily achieved by oral administration. At each dose level of verapamil tested, the combination with quinine proved more effective than either drug individually in reversing resistance to doxorubicin and vinblastine and synergistic chemosensitizing interaction was observed. Verapamil at 0.1 microgram/mL combined with quinine was capable of restoring sensitivity to doxorubicin fully and reduced resistance to vinblastine as effectively as verapamil alone at 1.0 micrograms/mL. Furthermore, the combination of 1.0 mumol verapamil with 10 mumols quinine increased accumulation and retention of anthracycline in the resistant cells to a greater extent than did either drug individually (P less than .001) and inhibited drug efflux as effectively as verapamil alone at 10 mumols. Our findings suggest that combined chemosensitization with verapamil and quinine may prove useful for overcoming MDR1 in patients with drug-refractory B-cell neoplasms such as multiple myeloma or non-Hodgkin's lymphomas.

Two human tumor cell lines exhibiting acquired multidrug resistance (MDR) with increased expression of a cell surface glycoprotein (GP-170) were tested for their sensitivity to human recombinant tumor necrosis factor (rTNF). The drug resistant mutant lines (CEM/V, a T-cell leukemia line resistant to vinblastine, and 8226/D, a multiple myeloma line resistant to doxorubicin), were markedly more sensitive to rTNF in clonogenic assay than were their drug-sensitive parental lines (CEM, 8226). As determined by radioreceptor assay, the number of cell surface receptors for rTNF did not differ on the parental and drug-resistant lines. During the first 24 hours after addition of rTNF, there was a decrease in intracellular ATP content in the CEM/V line but not in the CEM line. No differential effect of rTNF on ATP content was observed between 8226 and 8226/D. As determined by RNA dot-blot analysis, total cellular RNA for GP-170 was increased in the 8226/D cells. After rTNF exposure, expression of total cellular RNA for GP-170 was not altered. Accumulation of radiolabeled doxorubicin by 8226/D cells was not altered by previous or coincubation with rTNF. These findings suggest that the effects of rTNF on MDR cells is not related to TNF receptor number and is mediated at a step subsequent to rTNF binding and not by either inhibition of synthesis of GP-170 or by alteration in the function of the GP-170 efflux pump.

Using several multiple drug-resistant human myeloma cell lines as standards, we developed an immunohistochemical staining technique and means of quantitating P-glycoprotein in individual myeloma cells. The level of staining intensity for P-glycoprotein in individual myeloma cells was quantitated by measuring the average optical density of each cell with a microscopic computerized cell analysis system. Using this system, we observed that the level of P-glycoprotein for individual cells within a cell population of known drug sensitivity was very homogeneous (coefficient of variation less than or equal to 13%). Analysis of cell lines with gradually increasing levels of multidrug resistance (8226/S, 8226/Dox6 and 8226/Dox40) demonstrated a close association between the level of resistance to doxorubicin, defined by the mean lethal dose (D0) and the amount of P-glycoprotein on individual cells determined by the optical density (r = 0.82, P less than 0.0005). Intracellular doxorubicin (DOX) accumulation in the individual cell lines was inversely related to the level of drug resistance expressed as D0. P-glycoprotein was also detected in the marrow-derived myeloma cells of patients with drug refractory disease using immunohistochemical staining. The amount of P-glycoprotein in the cells of one patient was directly compared to the amount found in the simultaneously stained standard cell lines (8226/Dox6 and 8226/Dox40) by comparing the optical densities for individual cells. Using this immunohistochemical technique to detect and quantitate P-glycoprotein in patient myeloma cells and comparing it to standard multidrug resistant myeloma cell lines may be of value in determining the contribution of P-glycoprotein to clinical drug resistance in patients with multiple myeloma.