Efflux kinetics and intracellular distribution of daunorubicin are not affected by major vault protein/lung resistance-related protein (vault) expression

Vaults may contribute to multidrug resistance by transporting drugs away from their subcellular targets. To study the involvement of vaults in the extrusion of anthracyclines from the nucleus, we investigated the handling of daunorubicin by drug-sensitive and drug-resistant non-small lung cancer cells, including a green fluorescent protein (GFP)-tagged major vault protein (MVP)-overexpressing transfectant (SW1573/MVP-GFP). Cells were exposed to 1 microm daunorubicin for 60 min, after which the cells were allowed to efflux the accumulated drug. No significant differences in daunorubicin efflux kinetics were observed between the sensitive SW1573 and SW1573/MVP-GFP transfectant, whereas the drug-resistant SW1573/2R120 cells clearly demonstrated an increased efflux rate. It was noted that the redistribution of daunorubicin from the nucleus into distinct vesicular structures in the cytoplasm was not accompanied by changes in the intracellular localization of vaults. Similar experiments were performed using mouse embryonic fibroblasts derived from wild-type and MVP knockout mice, which were previously shown to be devoid of vault particles. Both cell lines showed comparable drug efflux rates, and the intracellular distribution of daunorubicin in time was identical. Reintroduction of a human MVP tagged with GFP in the MVP(-/-) cells results in the formation of vault particles but did not give rise an altered daunorubicin handling compared with MVP(-/-) cells expressing GFP. Our results indicate that vaults are not directly involved in the sequestration of anthracyclines in vesicles nor in their efflux from the nucleus.     

Early resistance to therapy during induction in childhood acute lymphoblastic leukemia

Many patients with acute lymphoblastic leukemia (ALL) are not cured by current therapy because of the development of drug resistance. It is not clear when resistance develops during the growth of the leukemic clone and whether resistant cells are already present at diagnosis or develop later during treatment. Twenty-two uniformly treated children with ALL were studied throughout induction treatment. The size of the leukemic clone in blood and marrow was estimated by limiting dilution PCR analysis, using the rearranged immunoglobulin heavy chain gene as a molecular marker. The decline in the number of leukemic cells was biphasic in virtually all patients. For both marrow and blood, the logarithmic mean of the number of leukemic cells fell by approximately four orders of magnitude during the first 2 weeks, one order of magnitude during the third week, and not at all during the last two weeks of induction treatment. For marrow, the median of the fraction of leukemic cells in each patient that survived per week of treatment was 0.008 for the first 2 weeks, 0.12 for the third week, and 1.4 for the last 2 weeks; for blood, the corresponding figures were 0.003, 0.14, and 0.69, respectively. In individual patients, the results for marrow and blood showed good correlation. The biphasic decline of leukemic cell number suggests that most leukemic cells were sensitive to treatment and were rapidly killed, leaving behind a minor but substantial population of drug-resistant cells. The most likely explanation for this phenomenon is that these resistant cells were already present at diagnosis, their resistance having originated from genetic or epigenetic mutations during prior growth of the leukemic clone.     

Differential expression of the lung resistance-related protein/major vault protein in the histological compartments of nephroblastomas.

Nephroblastomas (Wilms' tumors) are curable with survival rates above 80%. Some tumors, however, fail to respond to therapy and those patients have a poor prognosis. In a search for molecular markers of drug resistance, we investigated the expression of lung resistance protein (LRP) in tissue samples from 32 children with nephroblastoma by means of immunohistochemistry. LRP is a human major vault protein (MVP) and is associated with multidrug resistance of tumors. LRP/MVP expression was found in the blastemal and epithelial compartments but to a significantly lesser extent in the stromal compartment of Wilms' tumors. Expression was generally heterogeneous with respect to staining intensity and percentage of positive cells. We found significant relationships between LRP/MVP expression and chemotherapeutic pre-treatment of tumors and tumor stage. The immunohistochemical results were validated with a real-time RT-PCR technique and a significant association between protein and mRNA expression was observed.     

Array-based analysis of gene expression in childhood acute lymphoblastic leukemia

Gene expression profiles of 10 children with acute lymphoblastic leukemia (ALL) were detected using cDNA arrays. Total RNAs were isolated from peripheral blood leukocytes of the patients at diagnosis. For detection of expression profiles we used Atlas Human Cancer cDNA Arrays (Clontech) with 588 genes. Although the study revealed variability of gene expression in many genes, we identified a number of genes with the same expression changes (up-regulation: PCNA, ERCC1; down-regulation: jun-B, BCL-2 related protein A1, CRAF-1, PBP) in most examined patients. Our objective was to identify genes that were differentially expressed in ALL and might contribute to development (and characterization) of the disease.     

Adhesion molecule expression, clinical features and therapy outcome in childhood acute lymphoblastic leukemia.

In view of the relevance of adhesion molecule expression for the mechanisms of homing, trafficking and spreading of malignant cells, we have investigated the expression of surface adhesion molecules in lymphoblasts from 57 acute lymphoblastic leukemia (ALL) cases and tried to correlate the adhesive phenotype with immunological typing, prognostic factors at diagnosis and clinical follow-up. Blasts from all cases expressed adhesion molecules at high rates. Beta1 integrin chain (CD18) was consistently found on blasts from most ALL cases: among integrins of the beta2 family. LFA-1 was detected in 58% of cases, in the virtual absence of other alpha chains. CD54 and CD58 were expressed in variable proportions by ALL blasts and CD44 was detected in the majority of the malignant cells, whereas the CD62L selectin was only present in 24% of cases. B-lineage ALL's displayed similar adhesion molecule phenotypes irrespective of maturational stages of the leukemic cells. We found a significantly reduced expression of beta2 alphaL integrins in the hybrid ALL cases (CD13 and/or CD33 positive). However, these cases did not show differences in clinical presentation and behaviour in comparison with patients of other groups. We did not find a significant correlation between adhesion molecule expression and well established risk factors (age, white blood cell count, central nervous system involvement, chromosomal abnormalities), with the exception of splenomegaly, that was significantly associated with CD18 expression. In the follow-up, no evidence of significant correlation between adhesive phenotype and adverse events such as leukemic relapse and death was found. In conclusion, although expression of adhesion molecules on lymphoblasts confirms the phenotypic heterogeneity of ALL, it appears that this is not relevant for the clinical aspects of the disease and for prognosis.     

The role of breast cancer resistance protein in acute lymphoblastic leukemia.

PURPOSE: Overexpression of the transporter ABCG2, also known as breast cancer resistance protein and mitoxantrone resistance protein, can confer resistance to a variety of cytostatic drugs, such as mitoxantrone, topotecan, doxorubicin, and daunorubicin. This study analyzes the ABCG2 expression and activity in 46 human de novo acute lymphoblastic leukemia B- and T-lineage (ALL) samples. EXPERIMENTAL DESIGN: ABCG2 expression was measured flow cytometrically with the BXP-34 monoclonal antibody. ABCG2 functional activity was determined flow cytometrically by measuring mitoxantrone accumulation in combination with the ABCG2 inhibitor fumitremorgin C (FTC). To determine a possible effect of the transporters P-glycoprotein and multidrug resistance-associated protein (MRP1 and MRP2) on mitoxantrone accumulation, the accumulation was investigated in the presence of the P-glycoprotein inhibitor PSC 833 and MRP inhibitor MK-571. The ABCG2 gene was sequenced to investigate the amino acid at position 482. RESULTS: In B-lineage ALL (n = 23), the median BXP-34:IgG1 ratio was higher, namely 2.4 (range, 1.7-3.7), than in T-lineage ALL (n = 23; 1.9; range, 1.2-6.6; P = 0.003). The addition of FTC to mitoxantrone treatment caused a median increase in mitoxantrone accumulation of 21% (range, 0-140%) in B-lineage ALL. In T-lineage ALL, this FTC effect was less pronounced (5%; range, 0-256%; P = 0.013). The influence of FTC on mitoxantrone accumulation correlated with ABCG2 protein expression (r = 0.52; P < 0.001; n = 43). The increase in mitoxantrone accumulation, when FTC was added to cells treated with both PSC 833 and MK-571, correlated with the ABCG2 expression in B-lineage ALL but not in T-lineage ALL. Sequencing the ABCG2 gene revealed no ABCG2 mutation at position 482 in patients who accumulated more rhodamine after FTC. CONCLUSIONS: This study shows that ABCG2 is expressed higher and functionally more active in B-lineage than in T-lineage ALL.     

Impaired expression of interleukin 2 receptors in childhood acute lymphoblastic leukemia

Peripheral blood mononuclear cells (PBMC) from acute lymphoblastic leukemia (ALL) patients were studied for the expression of interleukin 2 receptors (IL-2R). When stimulated with phytohemagglutinin (PHA) for 3 days, the percentage of p55 Tac positive cells and the number of high affinity IL-2R was decreased in on-therapy ALL patients at remission and returned to normal after cessation of the treatment. However, the percentage of p75 IL-2R positive PBMC was normal in all ALL patients at remission. It is well-known that there are several immunological abnormalities including T and natural killer (NK) cell dysfunctions in ALL patients undergoing maintenance chemotherapy. IL-2 facilitates T-cell growth and corrects defective NK activity. Taking this into account, there may be a poor utilization of autologously produced IL-2 because of the impaired expression of IL-2R, and this poor utilization of IL-2 may be related to the defective immunological functions in childhood ALL patients at remission who are receiving maintenance chemotherapy.     

Significance of CD66c expression in childhood acute lymphoblastic leukemia

Upon analyzing 696 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases, we identified the characteristics of CD66c expression. In addition to the confirmation of strong correlation with BCR-ABL positivity and hyperdiploid, we further observed that CD66c is frequently expressed in CRLF2-positive (11/15, p < 0.01 against chimeric gene-negative) as well as hypodiploid cases (3/4), whereas it is never expressed in ETV6-RUNX1, MLL-AF4, MLL-AF9, MLL-ENL, and E2A-PBX1-positive cases. Although the expression of CD66c itself is not directly linked to the prognosis, the accompanying genetic abnormalities are important prognostic factors for BCP-ALL, indicating the importance of CD66c expression in the initial diagnosis of BCP-ALL.     

Chemokine receptor expression and function in childhood acute lymphoblastic leukemia of B-lineage

Scanty information is available on chemokine receptor expression and function in childhood B-lineage acute lymphoblastic leukemia (ALL). Thirteen pro-B, 17 early pre-B, 12 pre-B, and 9 B-ALL/Burkitt lymphoma (BL) pediatric cases were tested for CXCR1 to CXCR5 and CCR1 to CCR7 expression. CXCR2, CXCR3, and CXCR4 were expressed in the majority of cases, while the other receptors were variably expressed or absent. CXCR4 mediated chemotaxis of all leukemic cell subtypes. Freshly isolated CCR7(+) early pre-B-ALL cells migrated to CCL19, whereas CCR7(+) pro-B- and pre-B-ALL cells were attracted by CCL19 only following culture with soluble recombinant CD40 ligand.     

Differences in DNA-fingerprints between remission and relapse in childhood acute lymphoblastic leukemia

DNA "fingerprint" (DNA-F) analysis, based on the polymorphism caused by numeric variations in the tandem repeats of minisatellite areas of the human genome, has a potential capacity to reveal even minor genomic changes. In this study we have applied DNA-F to the detection of residual disease in leukemia. In order to identify normal and leukemic cell populations, we used two molecular probes: Jeffrey's minisatellite probes and M13 wild type phage probe, which detect different sets of polymorphic fragments in the human genome. Comparison of varying minisatellite fragments between remission and relapse was performed by Southern blot hybridization in seven patients with acute lymphoblastic leukemia (ALL). The results suggest that Southern hybridization with DNA "fingerprint" probes can prove to be a sensitive method in the detection of minimal residual disease in ALL.     

A critical risk-benefit assessment argues against the use of anthracyclines in induction regimens for newly diagnosed childhood acute lymphoblastic leukemia.

Although anthracyclines are associated with significant cardiac toxicity and their benefit remains unclear, they are included in nearly all current protocols for the treatment of childhood acute lymphoblastic leukemia (ALL). Currently open trials from most major groups use anthracyclines in the induction phase for all high-risk patients and in the delayed intensification phase for all patients regardless of risk classification. Our review of published randomized studies reveals no benefit for the addition of anthracyclines to induction phase of childhood ALL regimens consisting of vincristine, prednisone, and L-asparaginase (VPL), with or without a delayed intensification phase. No randomized studies have evaluated the use of anthracyclines in the delayed intensification phase of therapy. Furthermore, studies of relapsed patients indicated no benefit for the addition anthracyclines to maintenance regimens. Recent evidence from preclinical studies suggests that a combination of VPL with an anti-CD19 immunotoxin is more effective than VPL plus anthracyclines combination. Accumulated evidence exists that anthracyclines are associated with late-onset cardiac morbidity in about 25% of childhood ALL and other cancer survivors, and about 5% develop overt heart failure, with some requiring cardiac transplantation. Anthracycline-induced cardiotoxicity in children has no safe dose threshold and all doses are likely to cause significant myocardial damage. New data suggests that a unique cardiac mitochondrial exogenous NADH dehydrogenase is responsible for the anthracycline-induced oxygen radicals damage to the heart, and that chelators currently evaluated may not prevent late-onset cardiotoxicity in children. In view of these findings we urge extreme caution in using anthracyclines as part of multimodality ALL treatment programs, and strongly recommend reevaluation of what should be considered the best induction regimen for high-risk childhood ALL.     

儿童急性白血病初治患者LRP和MRP的表达及其临床意义

背景与目的:有研究发现肺耐药相关蛋白(lungresistanceprotein,LRP)和多药耐药相关蛋白(multidrugresistance鄄associatedporotein,MRP)的表达与白血病患儿耐药有关,而且患儿耐药不是由于一种耐药机制引起的。本研究旨在观察LRP和MRP在不同类型小儿白血病初治时的表达情况及其与临床疗效的关系。方法:应用逆转录鄄聚合酶链反应法(RT鄄PCR)检测38例不同类型儿童急性白血病[27例急性淋巴细胞白血病(acutelymphoblastic,ALL),11例急性非淋巴细胞白血病(acutenonlymphocyticleukemia,ANLL)]及6名正常儿童LRP、MRP基因的表达,结合患儿化疗后的完全缓解(CR)率分析两种基因表达的意义。结果:38例患儿化疗后CR26例(68.4%)。38例患儿中LRP基因表达阳性11例(28.9%),表达阴性27例(71.1%),6例正常对照儿童LRP基因阴性。LRP基因阳性患儿的CR率低于阴性者(分别为27.3%和85.2%,P<0.05)。MRP阳性者21例,6例正常对照儿童MRP基因阴性。MRP基因阳性者的CR率低于阴性者(分别为47.6%和94.1%,P<0.05)。27例ALL患儿中LRP阳性5例,11例ANLL患儿中LRP阳性6例(分别为18.5%和54.5%,P<0.05)。27例ALL患儿中MRP阳性16例;11例ANLL患儿MRP阳性5例(分别为59.3%和45.5%,P>0.05),ALL和ANLL中MRP的表达无显著性差异。LRP基因阳性患者的MRP阳性率与LRP阴性者MRP阳性率无显著性差异(分别为28.6%和29.4%,P>0.05),说明LRP基因与MRP基因之间无相关性。结论:LRP基因及MRP基因阳性儿童急性白血病者病情重、化疗缓解率低,儿童ANLL相对于ALL化疗缓解率低可能与LRP的表达有关。     

New definition of remission in childhood acute lymphoblastic leukemia.

The extent of clearance of leukemic cells from the blood or bone marrow during the early phase of therapy is an independent prognostic factor in acute lymphoblastic leukemia (ALL). Several methods are available to measure the minimal residual disease (MRD) remaining after initial intensive chemotherapy. The most promising are flow cytometric detection of aberrant immunophenotypes and polymerase chain reaction analysis of clonal antigen-receptor gene rearrangements. When applied together, these techniques enable one to monitor MRD in virtually all cases of ALL. Patients who achieve an 'immunologic' or 'molecular' remission (ie leukemic involvement of <0.01% of nucleated bone marrow cells at the end of remission induction therapy) are predicted to have a better clinical outcome than patients whose remission is defined solely by morphologic criteria. In studies to date, patients with MRD at a level of 10(-2) or more at the end of induction have fared almost as poorly as those with > or =5% blast cells in the bone marrow (ie induction failures). Sequential monitoring of MRD can improve the clinical utility of risk assessment still further. Additional studies are needed to determine the critical levels of MRD at various times of treatment and whether therapeutic intervention based on MRD findings can improve clinical outcome.     

Prognostic impact of CD45 antigen expression in high-risk, childhood B-cell precursor acute lymphoblastic leukemia.

To evaluate the clinical implications of CD45 expression in acute childhood lymphoblastic leukemia (ALL), we measured the CD45 expression of blast cells from 133 untreated patients with childhood B-precursor ALL (n = 118) or T-ALL (n = 15). CD45 expression (> or = 20%) was detected in all 15 cases (100%) of T-ALL, and 101 cases (86%) of B-precursor ALL. In 122 cases, the fluorescence intensity of the CD45 expression was measured as a relative value; the ratio of average linear values (RALV) of CD45 on the blasts to that on CD3-positive T-lymphocytes from the same specimen. The expression was more intense in the T-ALL cases than in the B-precursor ALL cases (RALV, mean +/- SE: T-ALL 0.230 +/- 0.04 vs. pro-B ALL 0.150 +/- 0.012/pre-B ALL 0.153 +/- 0.019, p < 0.05). However, the intensity of the CD10, CD19, CD20 and CD34 antigen immunoreactivity did not correlate with the CD45 expression. Patients with hyperdiploidy (chromosome number > 50) showed significantly lower levels of CD45 expression than patients with t(1;19) or normal karyotypes (RALV, mean +/- SE: 0.081 +/- 0.022 vs. 0.133 +/- 0.03/0.143 +/- 0.019, p < 0.05). Other clinical features such as age, gender and WBC count did not correlate with CD45 expression. The prognostic implications of CD45 expression were studied in non-high-risk (low-risk + intermediate-risk) (n = 60) and high-risk patients (n = 52) with B-precursor ALL who had been treated with the risk-directed protocol of ALL-941 trial. Although CD45 expression did not correlate with the event-free survival (EFS) of the non-high-risk patients, there was a significant correlation between the expression levels and the EFS of the high-risk patients: the 3-year EFS rate of the CD45low group (n = 26, RALV = 0.017-0.132) was 88 +/- 7% versus the CD45high group (n = 26, RALV = 0.133-0.450) at 34 +/- 24% (p < 0.05). These results show that the levels of expression of the CD45 antigen on leukemic lymphoblasts are significantly correlated with the clinical features and prognosis of childhood ALL.     

Post-induction residual leukemia in childhood acute lymphoblastic leukemia quantified by PCR correlates with in vitro prednisolone resistance.

Most prognostic factors in childhood acute lymphoblastic leukemia (ALL) are informative for groups of patients, whereas new approaches are needed to predict the efficacy of chemotherapy for the individual patient. The residual leukemia following 4 weeks of induction therapy with prednisolone, vincristine, doxorubicin and i.t. methotrexate and the in vitro resistance to prednisolone, vincristine, and doxorubicin were measured in 30 boys and 12 girls with B (n = 34) or T lineage (n = 8) ALL. The residual leukemia was quantified after 2 (MRD-D15, n = 29) and 4 weeks (MRD-PI, n = 42) of induction therapy with a precise and reproducible clone-specific PCR technique. The median MRD-D15 and MRD-PI were 0.50% (75% range 0.0088.1%) and 0.014% (75% range 0.001-2.0%), respectively, and these levels correlated significantly (n = 29, rs = 0.75, P < 0.001). Both the MRD-D15 and the MRD-PI were related to the age of the patient (MRD-D15: rs= 0.48, P= 0.009; MRD-PI: rs = 0.45, P = 0.003). Patients with T lineage ALL had higher MRD-PI than those with B lineage ALL (median MRD-PI: 0.5% vs 0.01%, P = 0.05). The median LC50 (concentration lethal to 50% of cells) for prednisolone was 2.3 microg/ml (75% range 0.05-668). Both MRD-D15 and MRD-PI correlated significantly with the in vitro resistance to prednisolone (MRD-D15: rs = 0.41, P = 0.03; MRD-PI: rs = 0.39, P = 0.01); but not to in vitro vincristine or doxorubicin resistance. The correlations between MRD and in vitro prednisolone resistance were even more pronounced when B cell precursor and T cell leukemia were analyzed separately (B cell precursor ALL: MRD-PI vs prednisolone LC50: n = 33, rs = 0.47, P = 0.006; T cell ALL: MRD-PI vs prednisolone resistance: n = 8, rs = 0.84, P = 0.009). After a median follow-up of 5.0 years (75% range 3.2-6.9) eight patients have relapsed. All of the 21 patients with a MRD-PI < or =0.5% and a prednisolone LC50 < or =10 microg/ml have remained in remission whereas the 7 year event-free survival for the remaining 20 patients was 0.45 +/- 0.16 (P= 0.002) Prospective studies in childhood ALL are needed to clarify whether combined monitoring of in vitro drug resistance and residual leukemia early during chemotherapy could offer new ways to classify patients and stratify the intensity of therapy.     

Non-T, non-B childhood acute lymphoblastic leukemia. Correlation between cytochemical markers and first complete remission

The positivity for four cytochemical reactions, acid phosphatase (AcP), alpha-naphtyl acid acetate esterase (ANAE), beta-glucuronidase (BG), and N-acetyl beta-glucosaminidase (NABG) was correlated to first remission duration in 120 children affected with non-T, non-B acute lymphoblastic leukemia (ALL). The percentages of patients remaining in complete remission at 72 months were always higher for children whose blasts lacked these enzymatic reactions; however, a statistical difference was found only between BG+ and BG- ALL. It also appears that more complete enzymatic patterns of leukemic cells are associated with a poorer prognosis. The percentage of patients still in their first remission was 89% for leukemias with no cytochemical markers, 59% when one reaction was present, but less than 39% when two or more enzymes were detected in the blasts. It is noteworthy that the blasts of patients with more severe prognosis demonstrated a simultaneous positivity for AcP-ANAE or BG-NABG cytochemical reactions. The possible usefulness of these cytochemical markers to detect subsets of patients with different prognostic significance among non-T, non-B ALL is discussed.