Expression of multiple myeloma associated markers in bone marrow spicules using a novel immunohistochemical technique.

Immunohistochemistry (IHC) is an important tool used for diagnosis and prognosis of several hematological malignancies, and it frequently is used for quantitative and qualitative analysis of expression of different protein biomarkers in tissue sections. To understand the histopathological alterations in multiple myeloma (MM), IHC analysis of bone marrow (BM) biopsy is commonly used. Owing to the harsh decalcification process generally used for processing of bone marrow biopsies, however, protein epitopes occasionally are rendered unsuitable for IHC detection. We have developed a novel technique for processing BM spicule samples into a fibrin clot matrix that allows IHC detection of MM protein markers. This method does not require decalcification and results in a consistent, reliable assay. Using paired BM spicule-clot and BM core biopsies from patients diagnosed with multiple myeloma, we studied six MM related antibodies including kappa and lambda immunoglobulin light chains, CD56, CD138, CYR61 and DKK1.

Expression of vascular endothelial growth factor and its receptors in multiple myeloma and other hematopoietic malignancies.

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. The importance of angiogenic factors such as VEGF, while clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. Human hematopoietic tumor cell lines, representing multiple lineages and diseases, produce and secrete VEGF and express at least one of its two receptors. Exposure of human vascular endothelial cells to VEGF increased the expression of several hematopoietic growth factors known to be involved in myeloma including interleukin-6 (IL-6). Bone marrow samples from patients diagnosed with multiple myeloma were examined for expression of VEGF and its receptors. VEGF protein production was detected in malignant plasma cells from 78% of the myeloma patients studied. While expression of the Flt-1 and KDR receptors was not observed in the malignant plasma cells, both were markedly elevated in the normal marrow myeloid and monocytic cells surrounding the tumor. In bone marrow clot sections from normal allogeneic donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myelocytes, macrophages, and megakaryocytes. In vitro colony-forming assays using patient-derived material revealed that antibody neutralization of VEGF resulted in an inhibition of colony growth, whereas the addition of recombinant human VEGF stimulated colony formation. Neutralization of VEGF activity also suppressed the generation of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from bone marrow mononuclear cells. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through paracrine and perhaps autocrine mechanisms.

Vascular endothelial cell growth factor is an autocrine promoter of abnormal localized immature myeloid precursors and leukemia progenitor formation in myelodysplastic syndromes.

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. To delineate the potential role of VEGF in patients with myelodysplastic syndrome (MDS), VEGF protein and receptor expression and its functional significance in MDS bone marrow (BM) were evaluated. In BM clot sections from normal donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myeloid elements. However, monocytoid precursors in chronic myelomonocytic leukemia (CMML) expressed VEGF in an intense cytoplasmic pattern with membranous co-expression of the Flt-1 or KDR receptors, or both. In situ hybridization confirmed the presence of VEGF mRNA in the neoplastic monocytes. In acute myelogenous leukemia (AML) and other MDS subtypes, intense co-expression of VEGF and one or both receptors was detected in myeloblasts and immature myeloid elements, whereas erythroid precursors and lymphoid cells lacked VEGF and receptor expression. Foci of abnormal localized immature myeloid precursors (ALIP) co-expressed VEGF and Flt-1 receptor, suggesting autocrine cytokine interaction. Antibody neutralization of VEGF inhibited colony-forming unit (CFU)-leukemia formation in 9 of 15 CMML and RAEB-t patient specimens, whereas VEGF stimulated leukemia colony formation in 12 patients. Neutralization of VEGF activity suppressed the generation of tumor necrosis factor-alpha and interleukin-1beta from MDS BM-mononuclear cells and BM-stroma and promoted the formation of CFU-GEMM and burst-forming unit-erythroid in methylcellulose cultures. These findings indicate that autocrine production of VEGF may contribute to leukemia progenitor self-renewal and inflammatory cytokine elaboration in CMML and MDS and thus provide a biologic rationale for ALIP and its adverse prognostic relevance in high-risk MDS.

Cocaine injection and coxsackievirus B3 infection increase heart disease during murine AIDS.

Coxsackievirus initiates myocarditis especially in the immunologically deficient or immature. To test whether Coxsackievirus B3 (CVB3) induced pronounced cardiomyopathy during severe immune dysfunction of murine AIDS, female C57BL/6 mice were infected with LP-BM5 retrovirus and superinfected with CVB3. Some were also injected daily with cocaine hydrochloride in 0.9% saline solution (30 mg/kg) intraperitoneally, because cocaine also suppresses cellular immune response. Heart tissue was analyzed histopathologically. Mice experiencing concurrent retrovirus and Coxsackievirus infection had a high degree of cardiac lesions consistent with myopathy compared with findings in uninfected animals (p <.05). Cocaine injection during murine retrovirus infection greatly exacerbated the pathogenesis of Coxsackievirus infection. C57BL/6 mice, essentially resistant to Coxsackievirus-induced cardiomyopathy, became susceptible during the immune dysfunction in murine AIDS. This suggests that retrovirus infection causes conditions favoring Coxsackie-induced cardiac lesions. Interleukin (IL)-2 and IL-4 expression by splenocytes from the dually infected retrovirus and Coxsackievirus group showed no significant differences when the animals were also cocaine treated. However tumor necrosis factor TNF-alpha production was significantly decreased in dually infected retrovirus + Coxsackievirus mice treated with cocaine, compared with findings in various controls (p <.05).

Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies.

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis by acting as a potent inducer of vascular permeability as well as serving as a specific endothelial cell mitogen. The importance of angiogenic factors such as VEGF, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. We examined the expression of mRNA and protein for VEGF in 12 human hematopoietic tumor cell lines, representing multiple lineages and diseases, including leukemia, lymphoma, and multiple myeloma. Our results revealed that VEGF message was expressed in these cells and that the corresponding protein was secreted into the extracellular environment. Five of the 12 cell lines were also found to express the Flt-1 receptor for VEGF at a moderate to strong level, suggesting an autocrine pathway. When human vascular endothelial cells were exposed to recombinant human VEGF, there was an increase in the mRNA for several hematopoietic growth factors including macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 6. Plasma cells in the bone marrow from patients diagnosed with multiple myeloma were found to express VEGF, whereas both the Flt-1 and KDR high affinity VEGF receptors were observed to be markedly elevated in the normal bone marrow myeloid and monocytic cells surrounding the tumor. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through either a paracrine or an autocrine mechanism.

Evidence for cytoplasmic P-glycoprotein location associated with increased multidrug resistance and resistance to chemosensitizers.

A new human myeloma cell line, 8226/MDR10V, was selected from a P-glycoprotein-positive cell line, 8226/Dox40, in the continuous presence of doxorubicin and verapamil. MDR10V cells are 13-fold more resistant to doxorubicin and 4-fold more resistant to vincristine than the parent cell line, Dox40. Chemosensitizers are also less effective in reversing resistance in the MDR10V compared to the Dox40 cells. Despite higher resistance to cytotoxic agents, MDR10V expresses 40% less P-glycoprotein in the plasma membrane compared to Dox40; however, total cellular P-glycoprotein is the same in both cell lines. Confocal immunofluorescence microscopy shows 2.5-fold more P-glycoprotein in the cytoplasm of MDR10V cells as compared to Dox40 cells. The cytoplasmic location of P-glycoprotein in the MDR10V cells is associated with a redistribution of doxorubicin. In Dox40 cells, doxorubicin is concentrated in the nucleus, whereas in MDR10V cells, 90% of doxorubicin is found in the cytoplasm. In the presence of equivalent intracellular doxorubicin, there was a decrease in DNA-protein crosslinks in the MDR10V cell line compared to the Dox40 cell line. This finding is in agreement with the intracellular doxorubicin fluorescence studies showing less doxorubicin in the nuclei of MDR10V cells compared to Dox40 cells. Verapamil is less effective in increasing doxorubicin accumulation in the nuclei of MDR10V cells compared to Dox40 cells. Processing of P-glycoprotein from the endoplasmic reticulum to the medial Golgi was identical between the two cell lines as determined by endoglycosidase H sensitivity of newly sensitized P-glycoprotein. No mutations were found in MDR1 cDNA from MDR10V cells compared to Dox40 cells. These results suggest that resistance to chemosensitizing agents plus cytotoxic drugs is associated with a redistribution of P-glycoprotein from the plasma membrane to the cytoplasm, which in turn reduces the amount of cytotoxic drug reaching the nucleus.

Rapid automated combined in situ hybridization and immunohistochemistry for sensitive detection of cytomegalovirus in paraffin-embedded tissue biopsies.

The authors questioned whether an automated kinetic mode assay of combined cytomegalovirus (CMV) late viral message and immediate and early antigens might result in a more sensitive and timely CMV diagnosis relevant to speedy treatment in the transplant setting. Toward this end, two cohorts were studied using automated in situ hybridization (ISH) for CMV as well as immunohistochemistry (IHC). The first cohort of patients consisted of 19 cases that were histologically positive (CMV-associated cytopathic change). A second cohort consisted of 10 cases that were histologically negative, yet culture positive. From the first cohort of histologically positive cases, 100% were positive by both ISH and IHC run on separate slides. In the second cohort, CMV was detected overall in 70% of cases (50% by ISH alone and 30% by IHC alone). These results indicate that a combined assay of ISH and IHC can detect more cases than routine hematoxylin and eosin staining or either assay alone. In two illustrative cases, used to demonstrate the feasibility of combining ISH and IHC, the authors used a combined two-color assay (ISH and IHC) performed sequentially on the same slide. The combined assays resulted in colocalized single cell message and protein in some cells and demonstrated more positive cells overall (some positive by IHC alone, some by ISH alone, and some by both) than either assay alone. The combined dual color assay can be completed within 4 to 5 hours giving the prospect of a same day result, which is faster than shell vial technique with immunofluorescence (24 to 48 hours) or culture (7 to 14 days). This study demonstrates that combining CMV message and protein assays results in a more sensitive assay and, when carried out in the kinetic mode, allows a speedy result relevant to early anti-CMV therapy.

Development of an orthotopic SCID mouse-human tumor xenograft model displaying the multidrug-resistant phenotype.

Multiple myeloma is a plasma cell malignancy which is generally incurable in spite of a high initial response to chemotherapy. While animal models of myeloma are known, the recent developments of human xenografts in nude and SCID mice suggests a promising experimental model. The SCID model, in particular, holds promise because these animals readily accept hematopoietic and lymphoid transplantation and do not generally develop graft versus host reaction. We have developed two drug-resistant variants of the human multiple myeloma cell line ARH-77 by in vitro exposure to gradually increasing concentrations of doxorubicin (ARH-D60) or mitoxantrone (ARM-80). When injected into irradiated SCID mice, the ARH-D60 cell line grew in an orthotopic pattern with the development of osteolytic lesions. This is in contrast to the 8226/C1N human myeloma cell line which grows in a disseminated but nonorthotopic manner in the SCID mouse. Both the ARH-D60 and ARM-80 cell lines are resistant to doxorubicin and cross-resistant to mitoxantrone, vinca alkaloids, taxol and m-AMSA while maintaining sensitivity to antimetabolites and alkylating agents. Growth characteristics and cell cycle kinetics, including S-phase, were not altered in the resistant sublines. The ARH-D60 and ARM-80 cell lines both displayed a classic multidrug-resistance (MDR) phenotype which was partially reversed by the addition of verapamil. These two cell lines represent the first MDR human myeloma cell lines which have demonstrated an orthotopic growth pattern in the SCID mouse and thus may be of value in studying the pathophysiology of this disease.

P-glycoproteins and multidrug resistance.

Multidrug resistance represents a major obstacle in the successful therapy of neoplastic diseases. Studies have demonstrated that this form of drug resistance occurs both in cultured tumor cell lines as well as in human cancers. P-glycoprotein appears to play an important role in such cells by acting as an energy-dependent efflux pump to remove various natural product drugs from the cell before they have a chance to exert their cytotoxic effects. Expression of the MDR1 gene product has been associated with a poor prognosis in clinical studies. It has been demonstrated in the laboratory that resistance mediated by the P-glycoprotein may be modulated by a wide variety of compounds. These compounds, which include verapamil and cyclosporin, generally have little or no effect by themselves on the tumor cells, but when used in conjunction with antineoplastic agents, they decrease, and in some instances eliminate, drug resistance. Clinical trials to modulate P-glycoprotein activity are underway at the present time to determine if such strategies will be feasible. Although the P-glycoprotein is expressed in many cell lines and occurs in patient tumors, its expression is not a universal feature of multidrug resistance, suggesting that other mechanisms are operating.

Cardiotoxicity in the SCID mouse following administration of doxorubicin and cyclosporin A.

Multiple myeloma is a plasma cell malignancy which is generally incurable in spite of a high initial response to chemotherapy. Relapsing disease commonly heralds an increase in the incidence of drug resistance which is often mediated by the product of the MDR-1 gene, P-glycoprotein (Pgp). One approach to modulating drug resistance due to Pgp overexpression has involved the use of agents known as chemomodulators which inhibit its function. We have developed a human xenograft model of multiple myeloma using the SCID mouse to evaluate the efficacy and toxicities of new MDR-1 chemomodulators. Cyclosporin A (CsA) is a widely used immunosuppressant which has been demonstrated to be a potent inhibitor of Pgp in vitro at concentrations which are clinically achievable. Preliminary studies revealed an acute toxicity in our SCID model which was associated with the combination of CsA and doxorubicin, and which was not observed with either drug alone, nor with cremaphor, the vehicle for CsA. In the current study, non-tumor bearing SCID mice were dosed with doxorubicin or the combination of doxorubicin with cremaphor, verapamil or CsA. Animals were sacrificed and tissues harvested for morphologic examination and for HPLC analysis of doxorubicin levels. In all tissues examined, there was a marked increase in tissue levels of doxorubicin when combined with CsA. Results also revealed a higher incidence and severity of myocardial damage in those animals receiving the combination of doxorubicin and CsA than in those receiving other combinations. The elevations in tissue levels observed with doxorubicin and CsA may contribute to the acute toxicities observed in the SCID mouse model.

Severe combined immunodeficiency (SCID) mouse modeling of P-glycoprotein chemosensitization in multidrug-resistant human myeloma xenografts.

We have established a reproducible in vivo model of human multiple myeloma in the severe combined immunodeficiency (SCID) mouse using both the drug-sensitive 8226/S human myeloma cell line and the P-glycoprotein-expressing multidrug-resistant 8226/C1N subline. As demonstrated previously, the SCID mouse is well suited as a model for myeloma because: (a) human SCID xenografts are readily attained; (b) human myeloma xenografts are readily detected by their immunoglobulin secretion; and (c) differential therapy effects in drug-sensitive versus drug-resistant cell lines are readily demonstrable by monitoring mouse urinary human immunoglobulin output. In the current study, we have utilized this model to evaluate the in vivo efficacy of chemomodulators of P-glycoprotein-related multidrug resistance. In our initial experiments, doxorubicin alone was effective in treating the 8226/S human myeloma xenografts but had no effect on the drug-resistant 8226/C1N xenografts, in the absence of the chemosensitizing agent verapamil. In subsequent experiments, the combination of verapamil and doxorubicin resulted in both a decrease in human lambda light chain urinary excretion and an increase in survival of those animals bearing the 8226/C1N tumor. The median survival time of animals injected with 8226/C1N cells and subsequently treated with doxorubicin was 48.6 +/- 7 days, which compared to a survival of 89.6 +/- 18 days in animals receiving the 8226/S cell line and treated with doxorubicin alone (P < 0.001). When verapamil was added to the treatment regimen of those animals bearing the 8226/C1N xenografts, there was a 179% increase in their life span (P < 0.001), which corresponded with the observed decreased light chain in the urine. In animals receiving multiple courses of chemotherapy, an attenuated response to verapamil and doxorubicin was observed, in a manner analogous to the clinical setting of human drug-resistant myeloma escape from chemosensitivity. The SCID human myeloma xenograft model thus offers a means of evaluating the in vivo efficacy and potential toxicities of new therapeutic approaches directed against P-glycoprotein in multidrug-resistant human myeloma.

Multidrug resistance in the laboratory and clinic.

Multidrug resistance represents a major obstacle in the successful therapy of neoplastic diseases. Studies have demonstrated that this form of drug resistance occurs in cultured tumor cell lines as well as in human cancers. P-glycoprotein appears to play an important role in such cells by acting as an energy-dependent efflux pump to remove various natural-product drugs from the cell before they have a chance to exert their cytotoxic effects. Using the tools of molecular biology, studies are beginning to reveal the true incidence of multidrug resistance, as mediated by the MDR1 gene, in the clinical setting. It has been demonstrated, at least in the laboratory, that resistance mediated by P-glycoprotein may be modulated by a wide variety of compounds, including verapamil and cyclosporine A. These are compounds which, by themselves, generally have little or no effect on the tumor cells, but when used in conjunction with antineoplastic agents act to decrease, and in some instances eliminate, drug resistance. The mechanism(s) by which these agents act to reverse resistance is not fully understood. Clinical trials to modulate P-glycoprotein activity are now under way to determine whether such strategies will be feasible. The detection of the P-glycoprotein in patient samples is very important in the design of these studies, as it appears that drug-resistant cells lacking P-glycoprotein will be unaffected by agents such as verapamil. Clinical studies are needed in which patients are stratified into chemotherapy protocols based on levels of MDR1 mRNA or P-glycoprotein expression in the primary tumors. Several research areas have been identified that are important to the transfer of the discovery of the MDR1 gene and its protein product from the research laboratory to the clinical environment. There is an immediate need for comprehensive information on the prevalence and levels of expression of the human MDR genes and their protein products in human organs and tissues. Data are needed on P-glycoprotein levels in specific subpopulations (e.g., according to age, sex, race, and diet), and the study of the heterogeneity and variability of expression of P-glycoprotein in normal human tissues should be given high priority. Since early studies have indicated some successes in identifying patients with classic multidrug resistance who might be responsive to chemosensitization, it can be anticipated that clinical research will accelerate in this area. The next wave of clinical studies will provide clinical investigators with opportunities to develop and evaluate P-glycoprotein tests and correlate test results with clinical outcomes.

An in vivo model of human multidrug-resistant multiple myeloma in SCID mice.

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.

Prediction of response to drug therapy of cancer. A review of in vitro assays.

Cancer chemotherapy has witnessed a great deal of progress since the introduction of the nitrogen mustards in the 1940s. Unfortunately, individual patients with apparently identical tumour histologies do not always respond identically to the same drug regimen. Determining the sensitivity and resistance of an organism before treatment has been the standard of care in infectious diseases for many years, while in oncology treatment has been initiated according to tumour histology rather than the tumour's sensitivity to a given agent. Attempts to individualise therapy have been the goal of oncologists since the 1950s. Since that time a number of in vitro assays have been developed to predict therapeutic outcome prior to the start of therapy. In the 1970s, with the introduction of the human tumour stem cell assay, it was generally believed that oncology was on the threshold of entering an era of predictive in vitro chemosensitivity testing. Unfortunately, this assay was shown to have a number of technical drawbacks including the low plating efficiencies of many primary tumour samples which thus limits the percentage which can be evaluated, leaving us still at this threshold today. Several recent developments, such as the Kern assay, which measures inhibition of radioactive precursors into tumour cells in the presence of antineoplastic agents, ATP bioluminescence assays, and the fluorescent cytoprint assay offer the promise of rapid and sensitive results. Other assays, such as the tetrazolium-based MTT and the sulphorhodamine blue assay appear to hold more promise in the screening and evaluation of potential new agents in established tumour cell lines than for evaluating chemosensitivity of clinical specimens. However, before a particular assay can be considered as an in vitro test of chemosensitivity or resistance, controlled prospective studies must be carried out to validate the assay in a number of different tumour types.

Altered expression of P-glycoprotein and cellular adhesion molecules on human multi-drug-resistant tumor cells does not affect their susceptibility to NK- and LAK-mediated cytotoxicity.

Drug resistance has been associated with resistance to NK- and LAK-cell-mediated cytotoxicity. We evaluated this issue in human cell lines, using multiple myeloma cells (8226) and 2 multi-drug-resistant (MDR) sublines selected using doxorubicin (8226/Dox40) and mitoxantrone (8226/MR40). In parallel, we studied the human breast carcinoma cell line series MCF7, MCF7/D40 and MCF7/Mitox. Unlike the sensitive parental cell lines, all 4 sublines display MDR-patterns of resistance, with the P-glycoprotein pump (P-170) detected only in the doxorubicin-selected sublines. Flow cytometric and immunocytochemical analyses showed expression of cellular adhesion molecules ICAM-I and LFA-3, and MHC-Class-I (MCF7/D40 only), to be decreased in the doxorubicin-selected MDR-sublines, whereas expression of CD56 (Leu 19) was strongly up-regulated in 8226/Dox40. Lysis of P-170-positive MDR tumor cells by NK or LAK cells was, however, unaffected by these alterations, suggesting redundancy in effector:target-cell adhesion pathways. Mitoxantrone-selected tumor cells did not display P-170, nor did they show altered expression of cellular adhesion molecules. Their susceptibility to NK or LAK cytolysis was also unimpaired as compared to the parental cell lines. Clinically, these results imply that immunotherapeutic modalities aiming at increased natural killer functions deserve full consideration even in patients who have become refractory to further cytostatic drug treatment.

Different mechanisms of decreased drug accumulation in doxorubicin and mitoxantrone resistant variants of the MCF7 human breast cancer cell line.

We selected two drug resistant variants of the MCF7 human breast cancer cell line by chronic in vitro exposure to doxorubicin (MCF7/D40 cell line) and mitoxantrone (MCF7/Mitox cell line), respectively. The cell lines are similar in growth characteristics including doubling time, DNA synthetic phase and cell size. Resistance to mitoxantrone conferred only partial resistance to doxorubicin; whereas resistance selected for doxorubicin appeared to confer complete resistance to mitoxantrone. Both agents selected for cross resistance to the Vinca alkaloids. MCF7/D40 cells display a classic-multi-drug resistance phenotype with expression of P-glycoprotein, decreased drug accumulation relative to the parental line and reversal of drug accumulation and drug resistance by verapamil. MCF7/Mitox cells likewise display resistance to multiple drugs, but in contrast to MCF7/D40 cells do not express P-glycoprotein by immunoblot or RNA blot analysis. Net drug accumulation in MCF7/Mitox cells was decreased relative to the parental cells but there was no selective modulation of drug accumulation or in vitro drug resistance by the addition of verapamil. Efflux of mitoxantrone was enhanced in both the MCF7/D40 and MCF7/Mitox cell lines relative to the MCF7/S cell line. We conclude that the two drug resistant cell lines have different mechanisms of decreased drug accumulation.

Development and characterization of a melphalan-resistant human multiple myeloma cell line.

We present data describing a human myeloma cell line (8226/LR-5) selected for resistance to melphalan which exhibits a 7-fold level of resistance to melphalan and is partially cross-resistant to other bifunctional alkylators and X-irradiation. Melphalan resistance is relatively unstable with a decrease in resistance observed within 17 weeks in the absence of drug. The resistance observed in this cell line is not mediated by reduced intracellular melphalan accumulation. DNA interstrand cross-linking at equivalent intracellular drug accumulation is significantly reduced in the resistant subline. Whether this reduction is the result of a decrease in the formation of this lesion or to an increased rate of removal of the lesion remains to be determined. Growth characteristics and cell cycle kinetics, including S phase, were similar between sensitive and resistant cell lines. Intracellular nonprotein thiols were found to be significantly elevated in the resistant 8226/LR-5 cells; as cells revert or lose resistance, intracellular nonprotein sulfhydryl levels decline. Prior treatment of the cells with buthionine sulfoximine significantly reduced nonprotein sulfhydryl levels and enhanced melphalan cytotoxicity in both the sensitive and resistant cell lines. Thiols appear to play a role in mediating melphalan resistance.

Role of glutathione and its associated enzymes in multidrug-resistant human myeloma cells.

Multidrug resistance (MDR) is a phenomenon associated with the emergence of simultaneous cross-resistance to the cytotoxic action of a wide variety of structurally and functionally unrelated antineoplastic agents. The present study was undertaken to determine if 8226 human myeloma cells possessing the MDR phenotype had an increased ability to resist the intercalating drug doxorubicin (DOX) via glutathione-based detoxification systems. Glutathione S-transferase (GST) was isolated by affinity chromatography, and the enzyme activity was assessed using 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates. There was no difference in overall GST activity between the sensitive and resistant cells. Using a cDNA probe (pGTSS1-2) for the human placental, anionic GST isoenzyme, no overexpression of mRNA for this isoenzyme was noted in the resistant line. When glutathione peroxidase activity (GSH-px) was assessed using either H2O2 or cumene hydroperoxide as substrate, again there was no difference in enzyme activity. Non-protein sulfhydryl (NPSH) levels were found to be elevated significantly in the resistant 8226/DOX40 subline (19.2 +/- 0.1 nmol NPSH/10(6) cells) as compared to the drug-sensitive parental subline 8226/S (11.6 +/- 1.9 nmol NPSH/10(6) cells) (P less than 0.001). In addition, when the 8226/DOX40 cells were cultured in medium without doxorubicin, there was a consistent decline in NPSH values reaching a steady state identical to that of the 8226/S cells. However, the decrease in NPSH level was not accompanied by a change in the level of doxorubicin resistance as assessed by colony-forming assays. Depletion of glutathione by D,L-buthionine-S,R-sulfoximine had no effect on doxorubicin sensitivity in either subline. Thus, it appears that GSH-based detoxification systems are not causally involved in maintaining the MDR phenotype in 8226 human myeloma cells; rather they appear to comprise an epiphenomenon associated with the resistance selection procedure.