A parallel-arm phase I trial of the humanised anti-IGF-1R antibody dalotuzumab in combination with the AKT inhibitor MK-2206, the mTOR inhibitor ridaforolimus, or the NOTCH inhibitor MK-0752, in patients with advanced solid tumours.

BACKGROUND: Two strategies to interrogate the insulin growth factor 1 receptor (IGF-1R) pathway were investigated: vertical inhibition with dalotuzumab and MK-2206 or ridaforolimus to potentiate PI3K pathway targeting and horizontal cross-talk inhibition with dalotuzumab and MK-0752 to exert effects against cellular proliferation, angiogenesis, and stem cell propagation. METHODS: A phase I, multi-cohort dose escalation study was conducted in patients with advanced solid tumours. Patients received dalotuzumab (10 mg kg(-1)) and escalating doses of MK-2206 (90-200 mg) or escalating doses of dalotuzumab (7.5-10 mg kg(-1)) and MK-0752 (1800 mg) weekly. Upon maximum tolerated dose determination, patients with low-RAS signature, high-IGF1 expression ovarian cancer were randomised to dalotuzumab/MK-2206 versus dalotuzumab/ridaforolimus, whereas patients with high IGF1/low IGF2 expression colorectal cancer received dalotuzumab/MK-0752. RESULTS: A total of 47 patients were enrolled: 29 in part A (18 in the dalotuzumab/MK-2206 arm and 11 in the dalotuzumab/MK-0752 arm) and 18 in part B (6 in each arm). Dose-limiting toxicities (DLTs) for dalotuzumab/MK-2206 included grade 4 neutropenia and grade 3 serum sickness-like reaction, maculopapular rash, and gastrointestinal inflammation. For dalotuzumab/MK-0752, DLTs included grade 3 dehydration, rash, and diarrhoea. Seven patients remained on study for >4 cycles. CONCLUSIONS: Dalotuzumab/MK-2206 and dalotuzumab/MK-0752 combinations were tolerable. Further developments of prospectively validated predictive biomarkers to aid in patient selection for anti-IGF-1R therapies are needed.

Identification of cryptic sites of DNA sequence amplification in human breast cancer by chromosome microdissection.

We have performed microdissection of 16 putative homogeneously staining regions (hsrs) from nine different breast cancer cell lines in order to determine their chromosomal origin and composition. As expected, the most commonly amplified chromosomal band-region was 17q12 (containing ERBB2). However, regions not containing known oncogenes were also identified, including 13q31 (5/9 cases) and 20q12-13.2 (4/9 cases). The chromosomal composition of the integrated amplified DNA within each hsr was determined and in 13/16 cases (81%), hsrs were shown to be composed of two or more chromosomal regions. These studies shed light on the mechanism of formation of hsrs, and identify chromosomal regions likely to harbour genes amplified in breast cancer.

Bone marrow stromal cells prevent apoptosis of lymphoma cells by upregulation of anti-apoptotic proteins associated with activation of NF-kappaB (RelB/p52) in non-Hodgkin's lymphoma cells.

Stromal cells are an essential component of the bone marrow microenvironment that regulate or supports tumor survival. In this study we therefore studied the role of stromal cells in lymphoma cell survival. We demonstrated that adhesion of the B-cell lymphoma cell lines SUDH-4 and 10 to bone marrow stroma inhibited mitoxantrone-induced apoptosis. This adhesion-dependent inhibition of mitoxantrone-induced apoptosis correlated with decreased activation of caspases-8 and 9, and cleavage of caspase 3 and PARP. Electrophoretic mobility shift assays (EMSA) analysis demonstrated significantly increased NF-kappaB binding activity in lymphoma cells adhered to stroma cells compared to lymphoma cells in suspension. This DNA binding activity could be attributed to cell adhesion-mediated proteolysis of the NF-kappaB precursor, p100 (NF-kappaB2). This resulted in the generation of active p52, which translocated to the nucleus in complex with p65 and RelB. Coculture with stromal cells also induced expression of the NF-kappaB-regulated anti-apoptotic molecules, XIAP, cIAP(1) and cIAP(2). Inhibition of NF-kappaB significantly suppressed HS-5-induced protection against apoptosis in lymphoma cell lines as well as in primary lymphoma cells. Thus, bone marrow stroma protects B-cell lymphoma cells against apoptosis, at least in part through activation of NF-kappaB dependent mechanism involving up-regulation of NF-kappaB regulated antiapoptotic proteins. Consequently, this study suggests a new approach to decrease the resistance of lymphoma to chemotherapy.

Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene.

O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.

Prediction of doxorubicin resistance in vitro in myeloma, lymphoma, and breast cancer by P-glycoprotein staining.

Prior studies have shown that the P-glycoprotein is a cell membrane efflux pump that is quantitatively increased in expression in multidrug-resistant tumor cell lines. In this study, fresh tumor tissues from patients with multiple myeloma, malignant lymphoma, or metastatic breast cancer were studied immunohistochemically for P-glycoprotein expression and for in vitro sensitivity to doxorubicin. Twenty-six patients who were either previously untreated or in relapse after chemotherapy had tumor specimens submitted that could be evaluated in both assays. The testing was done independently and blindly in separate laboratories instead of our being provided relevant clinical data on the patients. Tumor cells from 12 of the 26 patients (46%) stained positively for P-glycoprotein. Fifteen of the 26 specimens (58%) exhibited drug resistance in vitro. Although only three (21%) of the 14 P-glycoprotein-negative tumors exhibited in vitro resistance to doxorubicin, all 12 fresh tumors that stained positively for P-glycoprotein were resistant to doxorubicin. The difference in frequency of intrinsic doxorubicin resistance between P-glycoprotein-negative and -positive tumors was highly significant (P less than .001). Similar trends were observed in each of the individual tumor categories and were statistically significant in myeloma and breast cancer. Four of the biopsy specimens that stained positively for P-glycoprotein and exhibited doxorubicin resistance were from patients who had not received prior cytotoxic chemotherapy. Similar conclusions were reached when results of drug sensitivity tests were ranked in relation to the median infective dose rather than by criteria based on correlations with clinical drug resistance. Our findings indicate that positive staining for P-glycoprotein associated with multidrug resistance predicts intrinsic cellular resistance of human cancers to doxorubicin. We anticipate that immunohistochemical staining for P-glycoprotein will prove useful in clinical oncology.

Systemic toxic effects associated with high-dose verapamil infusion and chemotherapy administration.

Aside from its more conventional uses as a cardiovascular drug, the calcium channel blocker verapamil has recently been added to chemotherapeutic regimens to reduce drug resistance in B-cell and other neoplasms that express the P-glycoprotein. We recently treated patients with continuous-infusion verapamil (0.15 mg/kg per hour to 0.60 mg/kg per hour) over a 5-day period in combination with continuous-infusion vincristine and doxorubicin plus oral dexamethasone. Seventy-one courses involving 35 hospitalized patients were prospectively studied for cardiovascular and other side effects. Cardiovascular side effects were observed most frequently and consisted of first-degree heart block, hypotension, sinus bradycardia, and junctional rhythms. We observed higher degree heart block, but the QRS interval remained narrow and the ventricular escape rate remained relatively normal. Effects on mean arterial pressure, heart rate, and PR interval were both time and dose related. Severe, symptomatic congestive heart failure was rarely observed. The most common noncardiovascular side effects were constipation, peripheral edema, and weight gain. All systemic toxic effects observed were easily treated or disappeared with either temporary or permanent discontinuation of the verapamil infusion or by a decrease in the dose of verapamil. We conclude that the cardiovascular side effects associated with continuous, high-dose intravenous verapamil therapy are significant and dose limiting but are rapidly reversible. Less cardiotoxic chemosensitizers are needed to reverse multidrug resistance in cancer.

Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines.

BACKGROUND: Human cancer cell lines grown in the presence of the cytotoxic agent mitoxantrone frequently develop resistance associated with a reduction in intracellular drug accumulation without increased expression of the known drug resistance transporters P-glycoprotein and multidrug resistance protein (also known as multidrug resistance-associated protein). Breast cancer resistance protein (BCRP) is a recently described adenosine triphosphate-binding cassette transporter associated with resistance to mitoxantrone and anthracyclines. This study was undertaken to test the prevalence of BCRP overexpression in cell lines selected for growth in the presence of mitoxantrone. METHODS: Total cellular RNA or poly A+ RNA and genomic DNA were isolated from parental and drug-selected cell lines. Expression of BCRP messenger RNA (mRNA) and amplification of the BCRP gene were analyzed by northern and Southern blot hybridization, respectively. RESULTS: A variety of drug-resistant human cancer cell lines derived by selection with mitoxantrone markedly overexpressed BCRP mRNA; these cell lines included sublines of human breast carcinoma (MCF-7), colon carcinoma (S1 and HT29), gastric carcinoma (EPG85-257), fibrosarcoma (EPF86-079), and myeloma (8226) origins. Analysis of genomic DNA from BCRP-overexpressing MCF-7/MX cells demonstrated that the BCRP gene was also amplified in these cells. CONCLUSIONS: Overexpression of BCRP mRNA is frequently observed in multidrug-resistant cell lines selected with mitoxantrone, suggesting that BCRP is likely to be a major cellular defense mechanism elicited in response to exposure to this drug. It is likely that BCRP is the putative "mitoxantrone transporter" hypothesized to be present in these cell lines.

Collateral sensitivity to nitrosoureas in multidrug-resistant cells selected with verapamil.

We have examined the effects of the nitrosoureas, streptozotocin (STZ) and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), on a human multiple myeloma cell line, RPMI 8226, and its drug-resistant variants. Cell lines selected for doxorubicin (DOX) resistance alone displayed a STZ and BCNU cytotoxicity profile similar to that of the parent cell line. In contrast, two of the drug-resistant variants selected with DOX plus verapamil, an agent which inhibits P-glycoprotein-mediated multidrug resistance, displayed a collateral sensitivity to STZ and BCNU. Verapamil was included in the selection protocol because it has been shown to inhibit the P-glycoprotein-mediated multidrug resistance phenotype and is now in clinical trials as a chemosensitizing agent. The collateral sensitivity to these nitrosoureas seen in the DOX plus verapamil-selected cell lines is due to the functional loss of a DNA repair molecule, O6-Methylguanine DNA methyltransferase (MGMT). The functional loss of MGMT is secondary to the loss of MGMT gene expression. The loss of MGMT gene expression is not due to loss or gross rearrangement of the MGMT-coding region. If this selection pressure applied in vitro reflects the in vivo situation, then new chemotherapeutic strategies may be devised to exploit this phenomenon. These cell lines will serve as useful models for delineating mechanisms which govern MGMT expression.

Direct relation of DNA lesions in multidrug-resistant human myeloma cells to intracellular doxorubicin concentration.

Using a human myeloma cell line selected for resistance to doxorubicin (8226/DOX), which expresses the multidrug resistance phenotype, we studied the effects of drug accumulation on DNA damage and cytotoxicity in multidrug-resistant cells. The resistant 8226 subline showed a decrease in [14C]doxorubicin accumulation as compared to the sensitive (8226/S) subline at all time points investigated. DNA alkaline elution techniques were used to examine the formation of single and double strand breaks and DNA-protein cross-links following exposure to doxorubicin in both sensitive and resistant sublines. When 8226/S and 8226/DOX40 cells were exposed to the same extracellular concentration of doxorubicin there was a difference in the quantity of DNA lesions occurring, with the 8226/DOX40 line exhibiting less DNA damage. However, when the extracellular concentration of drug was adjusted to yield equivalent intracellular concentrations these differences were removed and both lines exhibited the same degree of damage for all three DNA lesions. The same DNA lesions were also studied in isolated nuclei from sensitive and resistant cells. Such a model removes any confounding factors due to drug accumulation such as altered cytosolic distribution and/or metabolism of drug. We observed no difference in the formation of single or double strand breaks, or DNA-protein cross-links when the nuclei were exposed to the same concentration of doxorubicin. Results from colony-forming assays have shown that when resistant and sensitive 8226 cells were exposed to high concentrations of doxorubicin, there was a good correlation between DNA damage, drug accumulation, and cytotoxicity. This relationship did not hold for lower concentrations of doxorubicin, for which there was a lack of correlation between drug accumulation and cytotoxicity. Such findings may possibly be due to a prolonged retention of drug by the sensitive cell line as compared to the resistant cells. Further studies are under way to examine this possibility.

Cytogenetic and phenotypic analysis of a human colon carcinoma cell line resistant to mitoxantrone.

A human colon carcinoma cell line selected for a 21-fold resistance to mitoxantrone was cross-resistant to the anthracycline, doxorubicin, but not to the anthracene, bisantrene. A 2-fold resistance was observed with vinblastine, another drug associated with multidrug resistance. Net intracellular mitoxantrone and doxorubicin accumulation were decreased at 1 h for all dose levels in the resistant cell line compared to the sensitive cell line. Although the resistant cells were more resistant to mitoxantrone than doxorubicin, the net accumulation of mitoxantrone was only 19% less than the sensitive cell line; whereas doxorubicin accumulation was decreased by 49%. No significant difference between the sensitive and resistant cell lines was observed in the initial accumulation of mitoxantrone; however, the efflux of mitoxantrone was increased in the resistant cell line. Verapamil did not overcome the resistance to mitoxantrone and did not increase the net accumulation of drug. No alterations in the electrophoretic mobility of membrane proteins were observed. Using immunoblotting techniques, the resistant cell line did not express P-glycoprotein which is frequently observed for cells resistant to anthracycline antibiotics. Cytogenetic analysis showed a putative homogenously staining region on the short arm of chromosome 7 in the resistant cell line. The limited cross-resistant phenotype, lack of verapamil reversal, nondetection of P-glycoprotein, and cytogenetic evidence of gene amplification suggests the involvement of a novel drug-resistant gene associated with resistance to mitoxantrone.

Pharmacological and biological evidence for differing mechanisms of doxorubicin resistance in two human tumor cell lines.

The cellular pharmacology of doxorubicin resistance (DOXR) has most commonly been associated with decreased drug uptake, enhanced drug efflux, cross-resistance to multiple anticancer agents, and the overproduction of a Mr 170,000 cell surface glycoprotein (termed P-glycoprotein). In this study, the pharmacological and genetic characteristics of two newly derived human DOXR sublines were examined. These DOXR sublines were established following continuously increasing DOX exposure until a 222-fold resistant fibrosarcoma subline (HT1080/DR4) and a 285-fold resistant colon adenocarcinoma subline (LoVo/DR5) were developed. However, three major lines of evidence suggest that despite the similar selection strategy, the mechanism of DOXR differs significantly between these two cell lines. First, Western blotting using the C219 antibody specific to P-glycoprotein revealed the overexpression of the Mr 170,000 cell surface glycoprotein in LoVo DOXR cells but not in HT1080 DOXR cells. Second, LoVo DOXR cells are cross-resistant to vincristine, actinomycin D, colchicine, etoposide, and gramicidin D, but not to 1-beta-D-arabinofuranosylcytosine. In contrast, HT1080 DOXR cells display cross-resistance to vincristine, actinomycin D, vinblastine, and etoposide; however, they are not cross-resistant to gramicidin D, and show an increased (approximately 18-fold) cross-resistance to 1-beta-D-arabinofuranosylcytosine. Third, intracellular DOX accumulation (as measured by [14C]DOX at 1-h and high-performance liquid chromatography analysis) was decreased approximately 2.7-fold in LoVo DOXR cells and approximately 2.0-fold in HT1080/DR4 cells. However, while net accumulation studies in the presence of 5 micrograms/ml verapamil reversed DOXR to parental values in LoVo colon adenocarcinoma cells, it only minimally decreased DOX resistance (12.6%) in HT1080/DR4 cells. Efflux patterns of [14C]DOX were similar for the DOXR sublines with an approximately 50% decrease in DOX retention after 1 h when compared to their respective parental cell lines. Our results suggest that DOXR in LoVo/DR5 cells may result from overexpression of P-glycoprotein. In contrast, DOXR in HT1080/DR4 appears to be non-P-glycoprotein mediated and may be related to an alternative mechanism capable of altering drug efflux or differential drug binding.

Analysis of multidrug resistance-associated protein (MRP) messenger RNA in normal and malignant hematopoietic cells.

The multidrug resistance-associated protein (MRP) gene is a member of the ATP-binding cassette transporter gene superfamily and may be partially responsible for clinical drug resistance. Reverse transcriptase-polymerase chain reaction was used to measure MRP mRNA in normal hematopoietic cells from bone marrow and peripheral blood as well as patients with high risk acute myelocytic leukemia and multiple myeloma. All normal peripheral blood cells, regardless of cell lineage (CD4, CD8, CD14, CD15, CD19, CD56), expressed a similar basal level of MRP mRNA. Specimens from bone marrow containing mixed lineages also expressed a similar basal level of MRP expression. In patients with acute myelocytic leukemia, 10 of 12 (83%) of the specimens had detectable MRP mRNA, but the level of expression was similar to that of normal blood cells and low compared to a cell line known to overexpress MRP (H69/AR). All myeloma patients (12 of 12) had detectable MRP mRNA expression at levels comparable to normal peripheral blood and bone marrow cells. We conclude that MRP is commonly expressed in normal hematopoietic cells as well as certain hematopoietic malignancies. The therapeutic relevance of MRP expression is unknown, but these studies emphasize the importance of measuring MRP expression in normal cells as a point of reference and comparison for detection in malignant cells. We also recommend obtaining sequential specimens from patients, which may reveal an increased expression of MRP from baseline as the disease progresses and becomes resistant.

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.

Characterization of a new drug-resistant human myeloma cell line that expresses P-glycoprotein.

Multiple myeloma is a disease with a high initial chemotherapeutic response but virtually no cures due to emergence of drug resistance. A doxorubicin-resistant human myeloma cell line (8226/Dox) has been selected from the myeloma cell line RPMI8226 by continuously exposing cells to gradually increasing doses of doxorubicin. The resistant phenotype has been retained for over 2 months despite growth in drug-free medium. The resistant subline was cross-resistant to mitoxantrone, acronycine, etoposide, and vincristine. The 8226/Dox cell line remained sensitive to melphalan but acquired collateral sensitivity to dexamethasone. Intracellular doxorubicin accumulation, as measured by [14C]doxorubicin and high-performance liquid chromatography, was decreased by 54% at 1 h for 8226/Dox compared to the sensitive line. Efflux of doxorubicin was significantly greater in the resistant subline as compared to the sensitive parent cell line. Membrane analysis using immunoblotting techniques detected increased expression of the integral membrane protein P-glycoprotein (Mr 170,000) in the resistant subline. Cytogenetic analysis of 8226/Dox revealed a 7q-anomaly not seen in the parent cell line. No double minutes or homogeneously staining regions were observed. The drug sensitivity/resistance pattern of the resistant cell line correlates well with clinical observations indicating the potential of this cell line as a model for resistance in multiple myeloma.

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.

Multidrug resistance in mitoxantrone-selected HL-60 leukemia cells in the absence of P-glycoprotein overexpression.

A multidrug-resistant variant of the human HL-60 promyelocytic leukemia cell line (HL-60/MX2) has been isolated in vitro by subculturing these cells in progressively increasing concentrations of mitoxantrone. The MX2 cells are cross-resistant to etoposide, teniposide, bisantrene, dactinomycin, 4'-(9-acridinylamino)methanesulfon-m-anisidide, and the anthracyclines daunorubicin and doxorubicin but retain sensitivity to the Vinca alkaloids melphalan and mitomycin C. In addition, the MX2 cells display slight collateral sensitivity to bleomycin. Despite being 30-35-fold less sensitive to mitoxantrone, net [14C]mitoxantrone accumulation at 60 min was reduced by only 10% in the mitoxantrone-resistant cells compared to the parental line. Furthermore, at later time points, e.g., 120 and 180 min, mitoxantrone accumulation in the MX2 cells exceeded that in HL-60 cells by 8.5 and 6.4%, respectively. No significant differences were observed between the sensitive and resistant cell lines in the initial (first 60 s) accumulation of mitoxantrone, and only minor (3-6%) enhancement of mitoxantrone efflux was detected in the resistant cell type. Monoclonal antibodies to P-glycoprotein had no detectable reactivity with membrane vesicles from either the sensitive or resistant cell types as determined by standard immunoblotting techniques. The mitoxantrone-resistant cells displayed a reciprocal translocation [rcpt(1;3)-(q21;p23)] not found in the sensitive parent, but there were no demonstrable double minute chromosomes or homogeneous staining regions in cells from either line. Thus, these mitoxantrone-resistant human leukemia cells display many features which are atypical for the "classic" multidrug resistance phenotype and should provide a useful model for the study of multidrug resistance which is not mediated by P-glycoprotein.

Monitoring the expression profiles of doxorubicin-induced and doxorubicin-resistant cancer cells by cDNA microarray.

Drug resistance in cancer is a major obstacle to successful chemotherapy. Cancer cells exposed to antitumor drugs may be directly induced to express a subset of genes that could confer resistance, thus allowing some cells to escape killing and form the relapsed resistant tumor. Alternatively, some cancer cells may be expressing an array of genes that could confer intrinsic resistance, and exposure to cytotoxic drugs select for the survival of these cells that form the relapsed tumor. We have used cDNA microarray to monitor the expression profiles of MCF-7 cells that are either transiently treated with doxorubicin or selected for resistance to doxorubicin. Our results showed that transient treatment with doxorubicin altered the expression of a diverse group of genes in a time-dependent manner. A subset of the induced genes was also found to be constitutively overexpressed in cells selected for resistance to doxorubicin. This distinct set of overlapping genes may represent the signature profile of doxorubicin-induced gene expression and resistance in cancer cells. Our studies demonstrate the feasibility of obtaining potential molecular profile or fingerprint of anticancer drugs in cancer cells by cDNA microarray, which might yield further insights into the mechanisms of drug resistance and suggest alternative methods of treatment.

Verapamil reversal of doxorubicin resistance in multidrug-resistant human myeloma cells and association with drug accumulation and DNA damage.

Verapamil reversed resistance to doxorubicin in a human multiple myeloma cell line selected for multiple drug resistance. The drug-resistant cell line 8226/DOX40 is known to have reduced intracellular drug accumulation associated with the overexpression of P-glycoprotein when compared to the sensitive parent cell line 8226/S. Verapamil alone was minimally cytotoxic in both cell lines, but reversed doxorubicin resistance in a dose-related manner in 8226/DOX40. A similar dose-response relationship was observed for verapamil in increasing net intracellular doxorubicin accumulation. This increased net accumulation was secondary to block of enhanced doxorubicin efflux by verapamil from resistant cells. In contrast, verapamil did not alter initial doxorubicin accumulation over the first 60 s when incubated with resistant cells. Addition of verapamil to the 8226/DOX40 cells enhanced the formation of doxorubicin-induced DNA single strand breaks, double strand breaks, and DNA-protein cross-links. Verapamil had no effect on these lesions in the drug-sensitive cells. In addition, verapamil did not affect chemotherapeutic cytotoxicity or transport in the drug-sensitive cell line. Verapamil appears to reverse doxorubicin resistance in this human myeloma cell line by blocking enhanced drug efflux, leading to increased drug accumulation and enhanced DNA damage.

Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line.

Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.

Overexpression of a dominant-negative signal transducer and activator of transcription 3 variant in tumor cells leads to production of soluble factors that induce apoptosis and cell cycle arrest.

Gene therapy of B16 tumors with a dominant-negative signal transducer and activator of transcription (Stat3) variant, designated Stat3beta, results in inhibition of tumor growth and tumor regression. Although only 10-15% of the tumor cells are transfected in vivo, the Stat3beta-induced antitumor effect is associated with massive apoptosis of B16 tumor cells, indicative of a potent bystander effect. Here, we provide evidence that blocking Stat3 signaling in B16 cells results in release of soluble factors that are capable of inducing apoptosis and cell cycle arrest of nontransfected B16 cells. RNase protection assays using multi-template probes specific for key physiological regulators of apoptosis reveal that overexpression of Stat3beta in B16 tumor cells induces the expression of the apoptotic effector, tumor necrosis factor-related apoptosis-inducing ligand. These in vitro results suggest that the observed in vivo bystander effect leading to tumor cell growth inhibition is mediated, at least in part, by soluble factors produced as a result of overexpression of Stat3beta in tumor cells.