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Aberrant expression of homeobox genes has been described in primary leukemia blasts. We recently cloned a new cDNA, BP1, which is a member of the homeobox gene family. BP1 expression was investigated in bone marrow samples from acute myeloid leukemia (AML), acute T cell lymphocytic leukemia (ALL) and pre-B cell ALL. Expression levels of two apparent isoforms of BP1, DLX7 and DLX4, were measured in the same samples. They are weakly if at all detectable in normal bone marrow, PHA-stimulated T cells or B cells. BP1 RNA was highly expressed in 63% of AML cases, including 81% of the pediatric and 47% of the adult cases, and in 32% of T-ALL cases, but was not found in any of the pre-B ALL cases. Coexpression of BP1, DLX7 and DLX4 occurred in a significant number of leukemias. Our data, including co-expression of BP1 with c-myb and GATA-1, markers of early progenitors, suggest that BP1 expression occurs in primitive cells in AML. Analysis of CD34+ and CD34- normal bone marrow cells revealed BP1 is expressed in CD34- cells and virtually extinguished in CD34+ cells. Ectopic expression of BP1 in the leukemia cell line K562 increased clonogenicity, consistent with a role for BP1 in leukemogenesis. The presence of BP1 RNA in leukemic blasts may therefore be a molecular marker for primitive cells and/or may indicate that BP1 is an important upstream factor in an oncogenic pathway. [ABSTRACT FROM AUTHOR]

A key issue in the treatment of acute leukemia is the development of resistance to chemotherapeutic drugs. Several mechanisms may account for this phenomenon, including failure of the cell to undergo apoptosis in response to chemotherapy, or failure of the drug to reach and/or affect its intracellular target. This review focuses on the latter mechanism, and on intracellular drug transport resistance mechanisms in particular. Expression of the ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) has generally been reported to correlate with prognosis in acute myeloid leukemia (AML). Additionally, but more controversial, expression of the ABC transporter multidrug resistance protein (MRP) and the vault-transporter lung resistance protein (LRP) have been correlated with outcome in AML. Despite these findings, functional efflux assays indicate the presence of non-Pgp, non-MRP transporters in AML. Recently, a novel ABC transporter, breast cancer resistance protein (BCRP) was cloned and sequenced in our laboratory. Transfection and overexpression of BCRP in drug-sensitive cells confers drug-resistance to the cells. BCRP is a half-transporter, and may homodimerize or form heterodimers (with a yet unknown half-transporter) to produce an active transport complex. Relatively high expression of BCRP mRNA is observed in approximately 30% of AML cases, suggesting a potential role for this new transporter in drug resistance in leukemia. [ABSTRACT FROM AUTHOR]

Purpose: It has been recognized that enhanced antioxidant defenses can contribute to the resistance of cancer cells displaying multidrug resistance (MDR) that arises in conjunction with the overexpression of P-glycoprotein (Pgp). The purpose of this study was to determine if the defenses against oxidant stress in MDR human leukemia cells (HL-60/AR) that overexpress multidrug-resistance-associated protein (MRP), but not Pgp, contribute to the mechanism of drug resistance in this cell line.Methods: HL-60/AR cells were evaluated in comparison with wild-type cells with respect to sensitivity to the oxidants hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (t-BuOOH), the activities and amounts of the antioxidant enzymes catalase and glutathione peroxidase (GSH-Px), and the effects that manipulation of the activities of these enzymes may have on cellular sensitivity to the oxidants and to daunorubicin. We also evaluated the ability of the cells to generate daunorubicin semiquinone free radical as measured by electron spin resonance (ESR) spectroscopy.Results: HL-60/AR cells were > 10-fold resistant to the cytotoxic effects of the H2O2 or t-BuOOH as compared with parental, drug-sensitive HL-60 cells. This phenomenon could be attributed largely to elevated activity and protein levels of catalase in HL-60/AR cells. Furthermore, inhibition of catalase by 3-amino-1,2,4-triazole (AT) diminished the resistance of HL-60/AR to these oxidants by > 80% or > 50%, respectively. Despite these findings, AT was incapable of causing sensitization of HL-60/AR cells to the cytotoxic effects of daunorubicin. We found that the activity and amount of selenium-dependent glutathione peroxidase (GSH-Px) was no greater in HL-60/AR cells than in HL-60 cells. Cultivation of cells in selenium-deficient medium caused a marked reduction in GSH-Px activity in HL-60/AR cells and a profound inhibition of GSH-redox cycling manifested by a decrease in baseline hexose monophosphate shunt activity (HMPS) and markedly blunted stimulation of the HMPS by the oxidant t-BuOOH in both wild-type and resistant cells. These variations in GSH-Px activity and GSH-redox cycling, however, were not associated with an alteration in cellular sensitivity to daunorubicin. The failure of catalase inhibition or selenium manipulation of GSH-Px activity to affect daunorubicin cytotoxicity was not due to the inability of these cells to produce free-radical species of daunorubicin, since ESR studies revealed that the generation of daunorubicin semiquinone free radical by HL-60/AR cells was equal to and, in fact, 3-fold that obtained with HL-60 cells.Conclusions: In comparison with parental HL-60 cells, MRP-overexpressing HL-60/AR cells have demonstrable alterations in antioxidant defenses that are manifested by cellular resistance to the cytotoxic effects of H2O2 and t-BuOOH and by elevated protein levels and activity of catalase. Whether these alterations are epiphenomena or are related to overexpression of MRP remains to be determined. However, it does appear that the enhanced antioxidant defenses observed in HL-60/AR cells do not contribute to the resistance to daunorubicin manifested by this cell line. Although HL-60/AR cells generate daunorubicin semiquinone free radical to an extent equal to or greater than that observed in HL-60 cells, the failure of alterations in GSH-Px activity or inhibition of catalase to change the sensitivity of HL-60/AR cells to daunorubicin suggests that the cytotoxicity of daunorubicin in these cells in not mediated through H2O2 or other peroxide species detoxified by these enzymes. [ABSTRACT FROM AUTHOR]

To improve our understanding of the mechanism of 1-beta-D-arabinofuranosylcytosine (ara-C) incorporation into DNA, we investigated the physical properties (size, position of nucleoside incorporation) of small fragments of nascent DNA (nDNA) obtained by pH-step alkaline elution of intact HL-60 cells following their exposure to ara-C. In the pH-step alkaline elution procedure, the smallest fragments of nDNA elute at pH 11. Anion-exchange high-performance liquid chromatography (HPLC) of nDNA obtained by 1 h elution at pH 11.0 of lysed HL-60 cells revealed a preponderance of nDNA fragments ranging from 0.5 to 40 kb in control ([3H]-dThd-labeled) cells. Exposure of cells to ara-C (0.8-1 microM) resulted in a loss of the preponderance of radiolabel in fragments of 0.5-40 kb along with redistribution of the radiolabel (from [3H]-dThd or [3H]-ara-C) into smaller nDNA fragments (predominantly < 100 bases in length) as determined by HPLC. We used the ability of pH-step alkaline elution to provide these small nDNA fragments produced by ara-C to investigate the paradoxical behavior of ara-C as a chain terminator in cell-free DNA synthetic systems while being incorporated into an internucleotide position in intact cells. Following the digestion of purified nDNA with micrococcal nuclease and spleen phosphodiesterase II, the proportion of radiolabel in 3'-dNMP (indicating an internucleotide position) or free nucleoside (indicating a chain terminus position) was determined by reverse-phase HPLC. In digests of prelabeled genomic DNA, as expected, > 90% of the radiolabel from [14C]-dThd or [3H]-ara-C was found to exist in an internucleotide position (as determined by co-chromatography with authentic 3'-dTMP or 3'-ara-CMP). In contrast, digests of nDNA that eluted at pH 11.0 revealed a significantly higher proportion of radiolabel in the chain terminus position (29%-35%) when the nDNA was obtained from cells exposed to 1 microM [3H]-ara-C as compared with cells exposed to [3H]-dThd or [3H]-dCyd alone (< 10%). These data obtained from pH-step alkaline elution of intact cells suggest that by causing the inhibition of chain elongation while failing to inhibit the formation of new nDNA replication intermediates, ara-C exposure leads to the production of very small nDNA fragments. This relative chain-terminating effect of ara-C is most apparent in the small nDNA replication fragments that elute at pH 11.0.(ABSTRACT TRUNCATED AT 400 WORDS) [ABSTRACT FROM AUTHOR]

We have developed a pharmacokinetic/pharmacodynamic approach that integrates the disposition, cytotoxic activity and interaction of anticancer drugs. Fundamental to this approach is the measurement of the cytotoxicity, against a "target" cell line, of patient plasma collected at different times after administration of the anticancer agent(s). To illustrate this approach, we have studied the plasma cytotoxic activity (PCA), against HL-60 cells, of plasma from 11 acute myeloblastic leukemic patients treated with daunorubicin (DNR). Plasma, obtained before and serially for 24 h after DNR treatment, was assayed by HPLC for DNR and daunorubicinol (DNRol), its active metabolite. The corresponding observed PCA values (PCAobs) against HL-60 cells were also measured with a flow-cytometric cell-survival assay that we had developed previously. The pharmacodynamics, i.e. PCA, were co-modeled (dual Hill equation with an interaction term to allow synergism or antagonism) with the pharmacokinetics. The integration of the PCA profile provided the area under the observed PCA versus time curve (AUCobs). For each patient, we also generated an "interaction panel", by adding known amounts of DNR and DNRol to his or her pretreatment plasma. The corresponding cytotoxicities were measured, and then applied to the pharmacodynamic model. This provided a standard surface from which the PCA of each sample obtained after therapy was predicted (PCAprd), on the basis of assayed concentrations of DNR and DNRol in that sample. For plasma samples obtained after treatment, the model simultaneously fit all three outputs, i.e. PCA and DNR/DNRol concentration, very well. We observed substantial interpatient variability in HL-60 growth rate in medium containing patient pretreatment plasma, in DNR activity in pretreatment plasma, and in the in vitro activity (PCA) of plasma obtained after DNR treatment. We also compared the AUCprd to the AUCobs for each patient, and we identified a subset of 4/11 acute myeloblastic leukemic patients who had developed much more PCA after DNR administration that could be explained by the measured concentrations of DNR and DNRol. This may be due to unidentified active metabolites or to factors produced in the plasma in response to the treatment. This pharmacokinetic/pharmacodynamic model is promising to describe pharmacodynamics and interactions of anticancer drugs in cancer patients. [ABSTRACT FROM AUTHOR]

The glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with [35S]-methionine, NaB[3H4], phosphorus 32, or sodium iodide I 125. HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with [35S]-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography. HL-60/S cells labeled with NaB[3H4] yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4. In contrast, NaB[3H4]-labeled HL-60/AR cells showed 7-8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4. In addition, [3H]-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of [3H]-glucosamine than did the former. Following treatment with tunicamycin, [3H]-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged. Surface-membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220-95 kDa) and low-molecular-weight ranges (21 kDa). Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen-specific antibodies demonstrated no antigenic differences between HL-60/S and HL-60/AR cells. HL-60/S cells incubated in the presence of tunicamycin, an inhibitor of N-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL-60/AR cells. In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells. These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated. These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220-95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein. Furthermore, in HL-60/S cells, the multidrug-resistant phenotype is partially inducible by inhibition of N-linked glycosylation of cell-surface proteins. [ABSTRACT FROM AUTHOR]

ATP-binding cassette proteins comprise a superfamily of transporter proteins, a subset of which have been implicated in multidrug resistance. Although P-glycoprotein was described over 15 years ago, the recent expansion in the number of transporters identified has prompted renewed interest in the role of drug transporters in clinical drug resistance. These newly identified transporters include additional members of the MRP family, ABC2, and a new half-transporter, MXR/BCRP/ABCP1. This half-transporter confers high levels of resistance to mitoxantrone, anthracyclines, and the camptothecins SN-38 and topotecan. At 72 kDa, MXR localizes to the plasma membrane in cells which highly overexpress the protein either through gene amplification or though gene rearrangement. Future studies will be aimed at identifying an inhibitor, and attempting to translate recognition of this new transporter into a target for anticancer treatment.

Stimulated by support from an R25 grant from the National Cancer Institute, we assembled a multidisciplinary team to design, implement, evaluate, and institutionalize in our medical school curriculum a training program to enable all students to graduate with basic competency in palliative and end-of-life care. In the fall of 1994, we reviewed the medical curriculum extensively to determine the need and optimal sites for integration of new educational modules. The freshman and junior years were found most suitable for our purposes; hence, behavioral objectives targeted at the freshman and junior medical student as learner were designed for the domain of palliative and end-of-life care. By 1996, negotiations with the medical school curriculum committee and with individual course masters secured permission and time allotments to include new coursework as required elements. We integrated approximately 20 hours of required training into the medical school curriculum, accomplished by modules that utilized small group learning, didactic presentations, out-of-classroom assignments, and practicum/experiential teaching methods. Freshman year curriculum consisted of a 3-hour module that considers the role of the physician in end-of-life care. Junior year training included a 1-hour module on advance directives and a 16-hour classroom and practicum course on hospice and end-of-life care. Performance-based assessment revealed that the students achieved the behavioral objectives. Furthermore, the students perceived benefit from the training and concluded that palliative care education should be a required part of their medical school experience. In September 2000, the medical school's Clinical Years Committee officially designated the palliative and end-of-life care training modules a mandatory part of the curriculum, with satisfactory completion a requirement for graduation.

The breast cancer resistance protein (BCRP) gene, formally known as ATP-binding cassette transporter G2 (ABCG2) gene, encodes an ABC half transporter that causes resistance to certain cancer chemotherapeutic drugs when transfected and expressed in drug sensitive cancer cells. Here we report the organization of the BCRP gene, and the initial characterization of the BCRP promoter. We identified the genomic sequence of BCRP and its promoter by screening a human genomic lambda phage library, as well as a BAC library, and by searching the human genome database. The BCRP gene spans over 66 kb and consists of 16 exons and 15 introns. The exons range in size from 60 to 532 bp. The translational start site is found in the second exon. The first exon contains the majority of the 5' UTR. Promoter activity was characterized by a luciferase reporter assay using transient transfection of the human breast cancer cell line MCF7, and the human choriocarcinoma cell lines JAR, BeWo and JEG-3, which we find to have high endogenous expression of BCRP. The BCRP gene is transcribed by a TATA-less promoter with several putative Sp1 sites, which are downstream from a putative CpG island. The sequence 312 bp directly upstream from the BCRP transcriptional start site conferred basal promoter activity. The 5' region upstream of the basal promoter is characterized by both positive and negative regulatory domains.

In addition to pain, patients who are approaching the end of life commonly have other symptoms. Unless contraindicated, prophylaxis with a gastrointestinal motility stimulant laxative and a stool softener is appropriate in terminally ill patients who are being given opioids. Patients with low performance status are not candidates for surgical treatment of bowel obstruction. Cramping abdominal pain associated with mechanical bowel obstruction often can be managed with morphine (titrating the dosage for pain) and octreotide. Delirium is common at the end of life and is frequently caused by a combination of medications, dehydration, infections or hypoxia. Haloperidol is the pharmaceutical agent of choice for the management of delirium. Dyspnea, the subjective sensation of uncomfortable breathing, is often treated by titration of an opioid to relieve the symptom; a benzodiazepine is used when anxiety is a component of the breathlessness.

PBK/TOPK is a recently cloned serine/threonine kinase which is phosphorylated during mitosis. Earlier work indicated that this kinase is upregulated in a Burkitt's lymphoma cell line (GA-10). To determine whether PBK/TOPK is upregulated in other mitotically active neoplastic cell lines and tissues, Northern analysis was performed on a panel of malignant cell lines and on clinical samples from patients with leukemia or lymphoma. While PBK/TOPK mRNA was not detectable in normal peripheral blood cells and was weakly expressed in hyperplastic tonsillar B-cells, significantly higher levels of mRNA were detected in 8 Burkitt's lymphoma cell lines, 10 other neoplastic cell lines, and 2 clinical samples-one derived from a patient with ALL and a second derived from a patient with relapsed myeloma. In addition, Northern analysis of fetal tissues showed upregulated expression of PBK/TOPK in fetal kidney, lung, spleen, brain, and testis. These data suggest that PBK/TOPK expression is increased in highly proliferative malignant cells and during normal fetal development., ((c)2001 Elsevier Science.)

Physical symptoms other than pain often contribute to suffering near the end of life. In addition to pain, the most common symptoms in the terminal stages of an illness such as cancer or acquired immunodeficiency syndrome are fatigue, anorexia, cachexia, nausea, vomiting, constipation, delirium and dyspnea. Management involves a diagnostic evaluation for the cause of each symptom when possible, treatment of the identified cause when reasonable, and concomitant treatment of the symptom using nonpharmacologic and adjunctive pharmacologic measures. Part I of this two-part article discusses fatigue, anorexia, cachexia, nausea and vomiting. Fatigue is the most common symptom at the end of life, but little is known about its pathophysiology and specific treatment. Education of the patient and family is the foundation of treatment with the possible use of adjunctive psychostimulants. Anorexia and cachexia caused by wasting syndromes are best managed with patient and family education, as well as a possible trial of appetite stimulants such as megestrol or dexamethasone. For appropriate pharmacologic treatment, it is helpful to identify the pathophysiologic origin of nausea in each patient.

Breast cancer resistance protein (BCRP), an ABC half-transporter, is overexpressed in cancer cell lines selected with doxorubicin/verapamil, topotecan, or mitoxantrone. BCRP-overexpressing cells show cross-resistance to camptothecin derivatives such as irinotecan, SN-38 (the active metabolite of irinotecan), and topotecan. To test whether BCRP confers SN-38 resistance, we selected two SN-38 resistant sublines from PC-6 human small-cell lung cancer cells by SN-38, and then characterized these cells. Compared to PC-6 cells, the resistant sublines PC-6/SN2-5 and PC-6/SN2-5H were approximately 18- and 34-fold resistant, respectively. The intracellular SN-38 accumulation was reduced in the sublines, and BCRP mRNA was overexpressed in proportion to the degree of SN-38 resistance. These findings suggest that BCRP confers SN-38 resistance in the sublines. To confirm this hypothesis, PC-6/SN2-5 cells were transfected with antisense oligonucleotides complementary to portions of BCRP mRNA. The antisense oligonucleotides significantly suppressed BCRP mRNA expression, and enhanced SN-38 sensitivity in the subline. These data indicate that BCRP is directly involved with SN-38 resistance, by efflux transport of SN-38.

Because the activities of HER family members are elevated and/or aberrant in a variety of human neoplasms, these cell surface receptors are receiving increasing attention as potential therapeutic targets. In the present study, we examined the effect of combining the HER family tyrosine kinase inhibitor CI1033 (PD 183805) with the topoisomerase (topo) I poison 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, in a number of different cell lines. Colony-forming assays revealed that the antiproliferative effects of simultaneous treatment with CI1033 and SN-38 were synergistic in T98G glioblastoma cells and HCT8 colorectal carcinoma cells, whereas sequential treatments were additive at best. In additional studies examining the mechanistic basis for these findings in T98G cells, immunoblotting revealed that the inhibitory effects of CI1033 on epidermal growth factor receptor autophosphorylation were unaffected by SN-38. Likewise, CI1033 had no effect on topo I polypeptide levels, localization, or activity. Nonetheless, CI1033 markedly enhanced the number of covalent topo I-DNA complexes stabilized by SN-38 or the related agent topotecan (TPT). Analysis of intracellular SN-38 levels by high-performance liquid chromatography and intracellular TPT levels by flow microfluorometry revealed that CI1033 increased the steady-state accumulation of SN-38 and TPT by 9.4 +/- 1.9- and 1.8 +/- 0.2-fold, respectively. Further evaluation revealed that the initial rate of TPT uptake was unaffected by CI1033, whereas the rate of efflux was markedly diminished. Additional studies demonstrated that T98G and HCT8 cells express the breast cancer resistance protein (BCRP), a recently cloned ATP binding cassette transporter. Moreover, CI1033 enhanced the uptake and cytotoxicity of SN-38 and TPT in cells transfected with BCRP but not empty vector. Conversely, CI1033 accumulation was diminished in cells expressing BCRP, suggesting that CI1033 is a substrate for this efflux pump. These results indicate that CI1033 can modulate the accumulation and subsequent cytotoxicity of two widely used topo I poisons in cells that have no history of previous exposure to these agents.

We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored. Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol.

Nursing facilities (NF) are important sites for the care of dying patients. Curricula likely to improve end-of-life care are needed for NF physicians. To this end, a model medical school palliative care curriculum was modified for experienced NF physicians. Adult learning techniques were emphasized, as well as interactions likely to change physician behavior. Inclusion of the opinion leader, audit with feedback, use of consensus guidelines, and other techniques for changing physician behavior were included. Written materials to supplement the course were identified. This new program was pilot tested and improved. An initial audit of physician practices and survey of the NF staff, a half-day adult educational session, and follow-up with the NF medical director is suggested. This intervention should be tested to determine if it improves patient end-of-life care outcomes in this setting.

Cellular resistance to the antifolate methotrexate (MTX) is often caused by target amplification, uptake defects, or alterations in polyglutamylation. Here we have examined MTX cross-resistance in a human breast carcinoma cell line (MCF7/MX) selected in the presence of mitoxantrone, an anticancer agent associated with the multidrug resistance (MDR) phenotype. Examination of protein expression and enzyme activities showed that MCF7/MX cells displayed none of the classical mechanisms of MTX resistance. They did, however, exhibit an ATP-sensitive accumulation defect accompanied by reduced polyglutamylation. Although the kinetics of drug uptake was similar between parental and resistant cells, the resistant cells exhibited increased energy-dependent drug efflux. This suggested the involvement of an ATP-binding cassette (ABC) transporter. However, cells transfected with the breast cancer resistance protein (BCRP)-the ABC transporter known to be highly overexpressed in MCF7/MX cells and to confer mitoxantrone resistance (D. D. Ross et al., J. Natl. Cancer Inst. 91: 429-433, 1999)-were not MTX resistant, which suggested that this transporter is not involved in MTX cross-resistance. Moreover, members of the MRP protein family of transport proteins, which had previously been implicated in MTX resistance, were not found to be overexpressed in the MCF7/MX cells. Thus, our data suggest that a novel MTX-specific efflux pump may be involved in this unusual cross-resistance phenotype.

Breast cancer resistance protein (BCRP) is a novel member of the adenosine triphosphate-binding cassette superfamily of transport proteins. Transfection and enforced expression of BCRP in drug-sensitive cells confer resistance to mitoxantrone, doxorubicin, daunorubicin, and topotecan. We studied blast cells from 21 acute leukemia patients (20 acute myeloid leukemia, 1 acute lymphocytic leukemia) for the expression of BCRP mRNA using a quantitative reverse-transcription polymerase chain reaction assay. BCRP mRNA expression varied more than 1000-fold among the samples tested, with low or barely detectable expression in half of the samples. Seven samples (33%) had relatively high expression of BCRP mRNA. High expression of BCRP did not correlate strongly with high expression of P-glycoprotein, suggesting that BCRP may cause resistance to certain antileukemic drugs in P-glycoprotein-negative cases. High expression of BCRP mRNA is sufficiently frequent in AML to warrant more extensive investigations to determine the relation of disease subtype and treatment outcome to BCRP expression and function.

Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry. These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance. High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan. Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression. Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells. Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein. Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance. Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required.

Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (MDRI) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). The presence of BCRP has thus far been reported solely using mRNA data. In this study, we describe a BCRP-specific monoclonal antibody, BXP-34, obtained from mice, immunized with mitoxantrone-resistant, BCRP mRNA-positive MCF-7 MR human breast cancer cells. BCRP was detected in BCRP-transfected cells and in several mitoxantrone- and topotecan-selected tumor cell sublines. Pronounced staining of the cell membranes showed that the transporter is mainly present at the plasma membrane. In a panel of human tumors, including primary tumors as well as drug-treated breast cancer and acute myeloid leukemia samples, BCRP was low or undetectable. Extended studies will be required to analyze the possible contribution of BCRP to clinical multidrug resistance.

Fumitremorgin C (FTC) is a potent and specific chemosensitizing agent in cell lines selected for resistance to mitoxantrone that do not overexpress P-glycoprotein or multidrug resistance protein. The gene encoding a novel transporter, the breast cancer resistance protein (BCRP), was recently found to be overexpressed in a mitoxantrone-selected human colon cell line, S1-M1-3.2, which was used to identify FTC. Because the drug-selected cell line may contain multiple alterations contributing to the multidrug resistance phenotype, we examined the effect of FTC on MCF-7 cells transfected with the BCRP gene. We report that FTC almost completely reverses resistance mediated by BCRP in vitro and is a pharmacological probe for the expression and molecular action of this transporter.

Background: Over a period of four years the authors developed and integrated into the curriculum of their medical school training programs in palliative medicine. Critical required elements in the freshman year focus on attitudes and the physician's role in the care of terminally ill patients and their families. A 16-hour module has been designed to be a required element for junior students. It includes in-depth classroom and experiential training in palliative medicine. The results of the pilot of this module are presented., Methods: The module consisted of one four-hour half-day session for four consecutive weeks during the ambulatory block in internal medicine. The first half-day class included both lectures and small-group discussions. Pain management, management of non-pain symptoms, and recognition of and basic interventions in spiritual and psychosocial suffering were covered. Required out-of-classroom reading assignments were distributed. The second and third half days were experiential, during which the student, in the company of a hospice nurse, made palliative care evaluations of terminally ill hospice patients. The last half day was a classroom session where the students presented their palliative care plan(s) for the patient(s) they had encountered on half days 2 and 3 to an interdisciplinary team (IDT) of the other students, a hospice medical director, a social worker, a hospice nurse, and a chaplain. Student scores on a 60-item objective test and participation in the IDT meeting were the primary data sources used to evaluate student achievement of the course objectives., Results and Conclusions: The majority of students attained the course objectives. Student evaluations of the module were very positive, particularly with regard to the home visits and the need for this training. It is anticipated that the module will be required during the 1999-00 academic year, with each student's performance contributing to his or her final grade in junior medicine.

To enhance the teaching of students to assess quality of life of patients with serious disease, the AACE Palliative Cancer Education Section has developed a teaching module. The module, which focuses on four desired learning objectives, is to be used in an hour-long small-group session. The authors describe the development of the module, as well as its objectives, teaching method, evaluation, and future challenges.

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.

Clinical studies are currently in progress to evaluate functional modifiers of P-glycoprotein (Pgp), an efflux pump associated with resistance to cancer chemotherapy. However, the effects of these modifiers on a more recently discovered efflux pump, the multidrug resistance associated protein (MRP), have not yet been fully characterized. MRP is expressed in most human tissues and is overexpressed in several tumor types. For these reasons, we have investigated the effects of three prototype Pgp modifiers, which act by different modes on the function of Pgp, on the function of MRP in two MRP-overexpressing cell lines: UMCC/VP lung and MCF-7/VP breast cancer cells. Clinically optimal plasma levels of verapamil, cremophor, and PSC833 have been shown to completely block the function of Pgp in Pgp-over expressing cells. However, in the two MRP-over expressing cell lines, these modifiers only partially blocked the function of MRP and combinations of these optimal concentrations acted antagonistically. Similar antagonistic effects were seen with combinations of suboptimal concentration levels of these blockers, while these combinations resulted in synergistic effects in Pgp overexpressing cells. For two biophysical parameters measured at the plasma membrane, membrane fluidity and membrane potential, the effects of these modifiers were essentially similar in Pgp and MRP expressing cells. We suggest that the 170 kD Pgp and the 190 kD MRP glycoproteins, imbedded in the plasma membranes, respond differently to simultaneous effects of the investigated prototype resistance modifiers. These results also suggest that the identification of the specific mechanism of drug resistance is important for the selection of chemotherapeutic strategies to block the efflux pump on the cancer cell.

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.

This review paper will focus on the molecular biological, biochemical and biophysical aspects of the following three efflux pumps: P-glycoprotein (Pgp), Multidrug Resistance Protein (MRP) and Lung cancer Resistance-related Protein. Since suppression of the function of these pumps has clinical implications, novel approaches developed in our laboratory for blocking the function of Pgp and MRP are also discussed. The second part of this review will summarize the clinical significance and the results of clinical trials designed to suppress the effects of these efflux pumps.

Human breast carcinoma MCF-7/AdrVp cells display a novel multidrug resistance phenotype that is characterized by the overexpression of a 95-kDa membrane glycoprotein (p95) and by marked reduction in intracellular anthracycline accumulation, without overexpression of P-glycoprotein or the multidrug resistance protein MRP. p95 is also highly expressed in multidrug-resistant NCI-H1688 cells derived from a human small cell lung carcinoma. Deglycoslyated p95 from NCI-H1688 cells was isolated by two-dimensional gel electrophoresis and then digested with trypsin. The tryptic peptides were analyzed by mass spectrometry and microsequencing. These analyses identified p95 to be identical to NCA-90, the nonspecific cross-reacting antigen related to the carcinoembryonic antigen (CEA). Further confirmation that p95 is indeed NCA-90 was obtained by Northern and Western blot studies using probes or antibodies specific for p95, NCA-90, or CEA family members. Western blot studies also revealed that CEA itself is overexpressed in MCF-7/AdrVp cells compared to parental MCF-7/W cells. The enforced expression of NCA-90 protein in HeLa cells stably transfected with NCA-90 cDNA did not result in increased resistance of the transfected cells to daunorubicin or a decrease in daunorubicin accumulation in the transfected cells compared to cells transfected only with the expression vector. However, a recent report by H. Kawaharata et al. (Int. J. Cancer, 72: 377-382, 1997) of diminished accumulation, retention, and cytotoxicity of doxorubicin in EJNIH3T3 cells in which enforced expression of CEA was accomplished leaves open the possibility that the overexpression of CEA, possibly in combination with that of NCA-90, could account at least in part for the drug resistant phenotype displayed by MCF-7/AdrVp cells.