Biomedical subjects

C P Reynolds

Publications and source records attributed to C P Reynolds.

At least 19 recordsLinked to original sources

PubMed2002

Synergistic cytotoxicity of buthionine sulfoximine (BSO) and intensive melphalan (L-PAM) for neuroblastoma cell lines established at relapse after myeloablative therapy.

Patients with high-risk neuroblastoma (NB) initially respond to aggressive, alkylator-based therapy only to die from recurrent disease that is refractory to chemotherapy, including alkylating agents. We examined the ability of buthionine sulfoximine (BSO)-mediated glutathione (GSH) depletion to modulate melphalan (L-PAM) resistance in five NB cell lines established after progressive disease following myeloablative therapy (high-dose melphalan, carboplatin, etoposide and total body irradiation) supported by autologous hematopoietic stem cell transplant (AHSCT), and in 15 NB cell lines established at diagnosis or after non-myeloablative therapy (pre-AHSCT). Four of five post-AHSCT NB cell lines and 10 of 15 pre-AHSCT NB cell lines were sensitive to single agent BSO (LC(90) <300 microM BSO), while two of five post-AHSCT lines and one of 15 pre-AHSCT lines showed high-level resistance to L-PAM (LC(90)>30 microM). Fixed ratio analysis demonstrated BSO/L-PAM synergy (combination index <1) for all five post-AHSCT and for all 15 pre-AHSCT cell lines tested. Multi-log cytotoxicity (often exceeding four logs of cell kill) was observed in post-AHSCT L-PAM-resistant cell lines (including p53 non-functional lines) only when clinically achievable concentrations of BSO were combined with myeloablative concentrations of L-PAM. We conclude that most neuroblastoma cell lines, including post-AHSCT NB cell lines that are highly resistant to myeloablative levels of L-PAM and lack p53 function, are sensitive to clinically achievable concentrations of L-PAM and BSO. However, some L-PAM-resistant NB cell lines (especially those lacking p53 function) require dose escalation of L-PAM to myeloablative concentrations in order to demonstrate significant synergistic cytotoxicity. Thus, optimal clinical application of BSO/L-PAM may require AHSCT.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2002

N-(4-hydroxyphenyl)retinamide increases ceramide and is cytotoxic to acute lymphoblastic leukemia cell lines, but not to non-malignant lymphocytes.

The retinoid, N-(4-hydroxyphenyl)retinamide (4-HPR), mediates p53-independent cytotoxicity and can increase reactive oxygen species and ceramide in solid tumor cell lines. We determined changes in ceramide and cytotoxicity upon treatment with 4-HPR (3-12 microM) in six human acute lymphoblastic leukemia (ALL) cell lines: T cell (MOLT-3, MOLT-4, CEM), pre-B-cell (NALM-6, SMS-SB), and null cell (NALL-1). Exposure to 4-HPR (12 microM) for 96 h caused 4.7 (MOLT-3), 3.5 (MOLT-4), 3.9 (CEM), 2.9 (NALM-6), 4.7 (SMS-SB), AND 4.5 (NALL-1) logs of cell kill. The average 4-HPR concentration that killed 99% of cells (LC(99)) for all six lines was 4.8 microM (range: 1.5-8.9 microM). Treatment with 4-HPR (9 microM) for 24 h resulted in an 8.9 +/- 1.0-fold (range: 4.9-15.7-fold) increase of ceramide. Ceramide increase was time- and dose-dependent and abrogated by inhibitors of de novo ceramide synthesis. Concurrent inhibition of ceramide glycosylation/acylation by d,l-threo-(1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol) (PPMP) further increased ceramide levels, and synergistically increased 4-HPR cytotoxicity in four of six ALL cell lines. 4-HPR was minimally cytotoxic to peripheral blood mononuclear cells and a lymphoblastoid cell line, and increased ceramide <2-fold. Thus, 4-HPR was cytotoxic and increased ceramide in ALL cell lines, but not in non-malignant lymphoid cell types.

Antineoplastic Agents↗

PubMed2001

Loss of p53 function confers high-level multidrug resistance in neuroblastoma cell lines.

Neuroblastomas can acquire a sustained high-level drug resistance during chemotherapy and especially myeloablative chemoradiotherapy. p53 mutations are rare in primary neuroblastomas, but a loss of p53 function could play a role in multidrug resistance. We determined p53 function by measuring induction of p21 and/or MDM2 proteins in response to melphalan (L-PAM) in seven L-PAM-sensitive and 11 L-PAM-resistant neuroblastoma cell lines. p53 was functional in seven/seven drug-sensitive but in only 4/11 drug-resistant cell lines (P = 0.01). In four of the seven cell lines lacking p53 function, mutations of p53 were detected by the microarray GeneChip p53 Assay and automated sequencing, whereas six cell lines with functional p53 had no evidence of p53 mutations. All of the cell lines with wild-type (wt) p53 showed a strong transactivation of the p53-HBS/CAT reporter gene, whereas the four cell lines with mutant p53 failed to transactivate p53 HBS/CAT. Overexpression of MDM2 protein (relative to p53 functional lines) was seen in two p53-nonfunctional cell lines with wt p53; one showed genomic amplification of MDM2. Nonfunctional and mutated p53 was detected in a resistant cell line, whereas a sensitive cell line derived from the same patient before treatment had functional and wt p53. Loss of p53 function was selectively achieved by transduction of human papillomavirus 16 E6 (which degrades p53) into two drug-sensitive neuroblastoma cell lines with intact p53, causing high-level drug resistance to L-PAM, carboplatin, and etoposide. These data obtained with neuroblastoma cell lines suggest that the high-level drug resistance observed in some recurrent neuroblastomas is attributable to p53 mutations and/or a loss of p53 function acquired during chemotherapy. If confirmed in patient tumor samples, these data support development of p53-independent therapies for consolidation and/or salvage of recurrent neuroblastomas.

Antineoplastic Agents↗

PubMed2001

N-(4-hydroxyphenyl)retinamide elevates ceramide in neuroblastoma cell lines by coordinate activation of serine palmitoyltransferase and ceramide synthase.

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR; fenretinide) is cytotoxic to a variety of cancer cell lines, and we previously showed an association between ceramide generation and 4-HPR cytotoxicity for neuroblastoma cell lines (B. J. Maurer et al., J. Natl. Cancer Inst. (Bethesda), 91: 1138-1146, 1999). Here we determine whether the increased ceramide mediated by 4-HPR in the CHLA-90 human neuroblastoma cell line results from de novo ceramide synthesis. Treatment of CHLA-90 with 4-HPR for 2 h, in the presence of [(3)H]palmitic acid, caused sequential formation of [(3)H]sphinganine (220% over control) and [(3)H]ceramide (160% over control), with sphinganine returning to baseline at 4 h, and ceramide continuing to increase (215% over control). 4-HPR treatment did not accelerate cellular decay of sphingomyelin. Preincubation of cells with either L-cycloserine, an inhibitor of serine palmitoyltransferase (SPT), or fumonisin B(1), an inhibitor of ceramide synthase, retarded ceramide formation in response to 4-HPR treatment, although sphinganine was still generated when 4-HPR and FB(1) were present. Data from in vitro enzyme assays using microsomes showed that preexposure of intact cells to 4-HPR resulted in a time (175% over control; 6 h)- and dose-dependent increase (173% over control; 10 microM) in SPT activity as well as a time (265% over control)- and dose-dependent increase (215% above control; 10 microM) in ceramide synthase activity. Our results show that 4-HPR-mediated ceramide generation is derived from the de novo synthetic pathway by coordinate activation of SPT and ceramide synthase. Knowledge of these biochemical events is of utility when downstream modulators of ceramide metabolism are used to heighten the cytotoxic response to chemotherapy.

Acyltransferases↗

PubMed2001

Homozygous deletion of CDKN2A (p16INK4a/p14ARF) but not within 1p36 or at other tumor suppressor loci in neuroblastoma.

Loss of heterozygosity of several specific genomic regions is frequently observed in neuroblastoma tumors and cell lines, but homozygous deletion (HD) is rare, and no neuroblastoma tumor suppressor gene (TSG) has yet been identified. We performed a systematic search for HD, indicative of a disrupted TSG, in a panel of 46 neuroblastoma cell lines. An initial search focused on a well-characterized consensus region of hemizygous deletion at 1p36.3, which occurs in 35% of primary neuroblastomas. Each cell line was screened with 162 1p36 markers, for a resolution of 13 kb within the consensus 1p36.3 deletion region and 350 kb throughout the remainder of 1p36. No HDs were detected. This approach was expanded to survey 21 known TSGs, specifically targeting intragenic regions frequently inactivated in other malignancies. HD was detected only at the CDKN2A (p16INK4a/p14ARF) gene at 9p21 and was observed in 4 of 46 cell lines. The observed region of HD included all exons of both CDKN2A and the closely linked CDKN2B (p15INK4b) gene for cell lines LA-N-6 and CHLA-174, all exons of CDKN2A but none of CDKN2B for CHLA-179, and only 104 bp within CDKN2A exon 2 for CHLA-101. All four deletions are predicted to inactivate the coding regions of both p16INK4a and p14ARF. HD was observed in corresponding primary tumor samples for CHLA-101 and CHLA-174 but was not present in constitutional samples. These results suggest that for neuroblastoma, large HDs do not occur within 1p36, most known TSGs are not homozygously deleted, and biallelic inactivation of CDKN2A may contribute to tumorigenicity in a subset of cases.

Blotting, Southern↗

PubMed2001

Detailed molecular analysis of 1p36 in neuroblastoma.

BACKGROUND: Several lines of evidence es tablish that chromosome band 1p36 is frequently deleted in neuroblastoma primary tumors and cell lines, suggesting that a tumor suppressor gene within this region is involved in the development of this tumor. PROCEDURE: We analyzed the status of 1p36 in primary neuroblastomas and cell lines to define the region of consistent rearrangement. RESULTS: Loss of heterozygosity (LOH) studies of primary neuro blastomas identified allelic loss in 135 of 503 tumors (27%), with the smallest region of overlap (SRO) defined distal to D15214 (1p36.3). No homozygous deletions were detected at 120 loci mapping to 1p36.1-p36.3 in a panel of 46 neuroblastoma cell lines. A recently identified patient with neuroblastoma was found to have a constitutional deletion within 1p36.2-p36.3, and this deletion, when combined with the LOH results, defined a smaller SRO of one megabase within 1p36.3. We constructed a comprehensive integrated map of chromosome 1 containing 11,000 markers and large-insert clones, a high-resolution radiation hybrid (RH) map of 1p36, and a P1-artificial chromosome (PAC) contig spanning the SRO, to further characterize the region of interest. Over 768 kb (75%) of the SRO has been sequenced to completion. Further analysis of distal 1p identified 113 transcripts localizing to 1p36, 21 of which were mapped within the SRO. CONCLUSION: This analysis will identify suitable positional candidate transcripts for mutational screening and subsequent identification of the 1p36.3 neuroblastoma suppressor gene.

Alleles↗

PubMed2001

Ganglioside GM2/GD2 synthetase mRNA is a marker for detection of infrequent neuroblastoma cells in bone marrow.

GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-Cer (GM2)/GalNAcbeta1-4(NeuAcalpha2-8NeuAcalpha2-3)Galbeta1-4Glcbeta1-1Cer (GD2) synthetase [beta-1,4-N-acetyl-galactosaminyl transferase (GalNAc-T)] mRNA, which encodes a key glycosyltransferase for ganglioside GD2 synthesis, was assessed as a molecular marker for detecting metastatic neuroblastoma cells in bone marrow (BM). GalNAc-T mRNA expression by neuroblastoma cell lines (n = 15), primary untreated neuroblastoma tumors (n = 29), morphologically normal BM (n = 22), peripheral blood stem cells (n = 10) from patients with cancers other than neuroblastoma, and blood mononuclear cells from normal donors (n = 17) was assessed by using reverse transcriptase-polymerase chain reaction (RT-PCR) and electrochemiluminescence detection assay (RT-PCR/ECL). BM harvested from 15 neuroblastoma patients was tested before and after ex vivo immunomagnetic bead purging, and results were compared to immunocytological analysis of the same specimens. All neuroblastoma cell lines (mean, 653 x 10(3) ECL units) and primary tumors (mean, 683 x 10(3) ECL units) were positive for significant expression of GalNAc-T mRNA compared to normal blood and BM cells. The RT-PCR/ECL assay could detect GalNAc-T mRNA in 100 pg of total RNA, and in a mixture of one neuroblastoma cell among 10(7) normal BM or blood cells. Eight of 15 autologous BM cells harvested from patients with neuroblastoma had tumor cells detectable by immunocytology, and all 15 were positive for GalNAc-T mRNA. After ex vivo purging, none of the BM cells was immunocytology-positive, but six remained positive by the RT-PCR/ECL assay. GalNAc-T mRNA provides a specific and sensitive molecular marker for RT-PCR/ECL detection of infrequent neuroblastoma cells in BM.

Adolescent↗

PubMed2001

Retinoid therapy of childhood cancer.

In vitro studies that showed RA could cause growth arrest and differentiation of myelogenous leukemia and neuroblastoma led to clinical trials of retinoids in APL and neuroblastoma that increased survival for both of those diseases. In the case of APL, ATRA has been the drug of choice, and preclinical and clinical data support direct combinations of ATRA with cytotoxic chemotherapy. For neuroblastoma, a phase I study defined a dose of 13-cis-RA, which was tolerable in patients after myeloablative therapy, and a phase III trial that showed postconsolidation therapy with 13-cis-RA improved EFS for patients with high-risk neuroblastoma. Preclinical studies in neuroblastoma indicate that ATRA or 13-cis-RA can antagonize cytotoxic chemotherapy and radiation, so use of 13-cis-RA in neuroblastoma is limited to maintenance after completion of cytotoxic chemotherapy and radiation. A limitation on the antitumor benefit of ATRA in APL is the marked decrease in drug levels that occurs during therapy as a result of induction of drug metabolism, resulting in a shorter drug half-life and decreased plasma levels. Although early studies sought to overcome the pharmacologic limitations of ATRA therapy in APL, the demonstration that ATO is active against APL in RA-refractory patients has led to a focus on studies employing ATO. Use of 13-cis-RA in neuroblastoma has avoided the decreased plasma levels seen with ATRA. It is likely that recurrent disease seen during or after 13-cis-RA therapy in neuroblastoma is due to tumor cell resistance to retinoid-mediated differentiation induction. Studies in neuroblastoma cell lines resistant to 13-cis-RA and ATRA have shown that they can be sensitive, and in some cases collaterally hypersensitive, to the cytotoxic retinoid fenretinide. Fenretinide induces tumor cell cytotoxicity rather than differentiation, acts independently from RA receptors, and in initial phase I trials has been well tolerated. Clinical trials of fenretinide, alone and in combination with ceramide modulators, are in development.

Antineoplastic Agents↗

PubMed2001

Buthionine sulfoximine and myeloablative concentrations of melphalan overcome resistance in a melphalan-resistant neuroblastoma cell line.

BACKGROUND: Alkylator resistance contributes to treatment failure in high-risk neuroblastoma. Buthionine sulfoximine (BSO) can deplete glutathione and synergistically enhance in vitro sensitivity to the alkylating agent melphalan (L-PAM) for many neuroblastoma cell lines, but optimal use of this combination needs to be defined because clinical responses have been less frequent and not durable. PATIENTS AND METHODS: The authors established and characterized a neuroblastoma cell line (CHLA-171) from a patient who died of progressive disease after treatment with BSO and low-dose L-PAM. RESULTS: CHLA-171 lacks MYCN amplification, expresses PGP (P-glycoprotein) 9.5 RNA, and shows cell surface antigen expression (human leukocyte antigen class I weakly positive, but HSAN 1.2 (hybridoma, SAN 1.2) and anti-GD2 (anti-ganglioside GD2 antibody) strongly positive) characteristic of neuroblastoma cell lines. Twenty-four hours of BSO treatment (0-1,000 micromol/L) maximally depleted CHLA-171 glutathione to 36% of baseline. The cytotoxic response of CHLA-171 to BSO and L-PAM, alone and in combination, was measured by digital image microscopy (DIMSCAN) over a range of drug concentrations and compared with drug levels obtained in the patient during BSO/L-PAM therapy. As single agents, CHLA-171 was highly resistant to L-PAM (LD90 = 42 micromol/L; peak plasma concentration in the patient equals 3.9 micromol/L) and moderately resistant to BSO (LD90 = 509 micromol/L; steady-state concentration in the patient equals 397 micromol/L). Treatment with a 10:1 (BSO:L-PAM) fixed ratio combination synergistically overcame resistance (3-4 logs of cell kill, combination index <1) at clinically achievable levels of BSO (100-400 micromol/L) and levels of L-PAM (10-40 micromol/L) clinically achievable only with hematopoietic stem cell support. CONCLUSIONS: The in vitro results obtained for CHLA-171 suggest that BSO/L-PAM therapy may be optimally effective for drug-resistant neuroblastoma using myeloablative doses of L-PAM.

Antimetabolites, Antineoplastic↗

PubMed2000

Quantitative tumor cell content of bone marrow and blood as a predictor of outcome in stage IV neuroblastoma: a Children's Cancer Group Study.

PURPOSE: This study investigated the prognostic value of quantifying tumor cells in bone marrow and blood by immunocytology in children with high-risk, metastatic neuroblastoma. PATIENTS AND METHODS: Patients with stage IV neuroblastoma (N = 466) registered on Children's Cancer Group study 3891 received five cycles of induction chemotherapy and were randomized either to myeloablative chemoradiotherapy with autologous purged bone marrow rescue or to nonmyeloablative chemotherapy. Subsequently, they were randomized to 13-cis-retinoic acid or no further treatment. Immunocytologic analyses of bone marrow and blood were performed at diagnosis, week 4, week 12, bone marrow collection, and end induction and were correlated with tumor biology, clinical variables, treatment regimen, and event-free survival (EFS). RESULTS: Immunocytology identified neuroblastoma cells in bone marrow of 81% at diagnosis, 55% at 4 weeks, 27% at 12 weeks, 19% at bone marrow collection, and 14% at end induction. Tumor cells were detected in blood of 58% at diagnosis and 5% at collection. There was an adverse effect on EFS of increasing tumor cell concentration in bone marrow at diagnosis (P =.04), at 12 weeks (P =.006), at bone marrow collection (P <.001), and at end induction (P =.07). Positive blood immunocytology at diagnosis was associated with decreased EFS (P: =.003). The prognostic impact of immunocytology was independent of morphologically detected bone marrow disease, MYCN status, and serum ferritin level in bivariate Cox analyses. CONCLUSION: Immunocytologic quantification of neuroblastoma cells in bone marrow and blood at diagnosis and in bone marrow during induction chemotherapy provides prognostic information that can identify patients with very high-risk disease who should be considered for experimental therapy that might improve outcome.

Adolescent↗

PubMed2000

Synergistic cytotoxicity in solid tumor cell lines between N-(4-hydroxyphenyl)retinamide and modulators of ceramide metabolism.

BACKGROUND: We previously reported that N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) treatment caused large increases of ceramide levels in neuroblastoma cell lines and induced cell death by a combination of apoptosis and necrosis through p53 (also known as TP53)-independent and caspase-independent pathways. Our goal was to determine if several molecules that inhibit enzymes involved in ceramide metabolism-L-threo-dihydrosphingosine (safingol), d, l-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), and tamoxifen-enhanced 4-HPR-mediated cytotoxicity and/or affected ceramide levels. METHODS: Cellular lipids were quantified by radiolabeling and thin-layer chromatography. Cytotoxicity and cytotoxic synergy (expressed as combination index, where combination index <1 indicates synergy and >1 indicates antagonism) were measured in cultured cancer cell lines with the use of a fluorescence-based assay of cell viability employing digital imaging microscopy. Statistical tests were two-sided. RESULTS: 4-HPR increased ceramide levels by de novo synthesis. Safingol (1-4 microM) was incorporated into a stereochemical variant of ceramide and synergized with a 3:1 molar ratio of 4-HPR (3-12 microM), to produce a 100-fold to 10 000-fold (2 to 4 logs) increase in cytotoxicity relative to 4-HPR alone in neuroblastoma (combination index <0.1), lung (combination index <0.1-0.2), melanoma (combination index <0.1-0.2), prostate (combination index <0.1-1.0), colon (combination index 0.1-0.3), breast (combination index = 0.1-0.5), and pancreas (combination index = 0.2) cell lines, including p53 mutant and alkylator-resistant cell lines. The 4-HPR and safingol combination was cytotoxic in low-oxygen conditions and was minimally toxic to normal fibroblasts and bone marrow myeloid progenitor cells. Addition of agents that retard ceramide glucosylation and/or acylation, such as PPMP or tamoxifen, to 4-HPR or to the combination of 4-HPR and safingol further increased cytotoxicity to tumor cells. CONCLUSIONS: Combinations of 4-HPR and modulators of ceramide metabolism may form the basis for a novel chemotherapy that is functional under hypoxic conditions (e.g., such as those within tumors) and is p53 independent and caspase independent.

Antineoplastic Agents↗

PubMed2000

p53 mutations and loss of p53 function confer multidrug resistance in neuroblastoma.

BACKGROUND: Neuroblastomas often acquire sustained drug resistance during therapy. Sensitivities to carboplatin, etoposide, or melphalan were determined for 18 neuroblastoma cell lines; eight were sensitive and ten were resistant. As p53 mutations are rare in neuroblastomas studied at diagnosis, we determined if acquired p53 mutations and loss of function conferred multidrug resistance. RESULTS: Loss of p53 function (p53-LOF), defined as a failure to induce p21 and/or MDM2 in response to melphalan, was seen in 1/8 drug-sensitive and 6/10 drug-resistant cell lines. In four cell lines p53-LOF was associated with mutations in the DNA binding region of p53, while three cell lines with LOF and four cell lines with functional p53 had no evidence of p53 muta-tions. Nonfunctional and mutated p53 was detected in one resistant cell line, while a sensitive cell line derived from the same patient prior to treatment had functional and wild type (wt) p53. We transfected HPV 16 E6 (which mediates degradation of p53, causing LOF) into two drug-sensitive neuroblastoma cell lines with functional p53. LC(90) values of HPV 16 E6 transfected cell lines were 3-7-fold (melphalan), 8-109-fold (carboplatin), and 2-158-fold (etoposide) greater than that of LXSN-transfected controls. CONCLUSIONS: These data suggest that some neuroblastomas acquire p53 mutations during therapy, which is associated with a loss of p53 function, and can confer high-level multidrug resistance.

Chromosome Deletion↗

PubMed2000

Retinoic-acid-resistant neuroblastoma cell lines show altered MYC regulation and high sensitivity to fenretinide.

BACKGROUND: High-dose, pulse-13-cis-retinoic acid (13-cis-RA) given after intensive cytotoxic therapy improves event-free survival for high-risk neuroblastoma (NB), but more than 50% of patients have tumor recurrence. PROCEDURE: We conducted multistep selection for resistance to all-trans-retinoic acid (ATRA) in NB cell lines with (SMS-KCNR and LA-N-5) or without (SMS-LHN) MYCN genomic amplification. RESULTS: After 12 exposures to 10 microM ATRA, the two MYCN-amplified cell lines (KCNR 12X RR and LA-N-5 12X RR) showed partial resistance to the cytostatic/differentiation effects of ATRA; complete resistance was seen in LHN 12X RR. ATRA-selected cells showed general RA resistance (cross-resistance to 13-cis-RA). Transient (KCNR 12 X RR, LA-N-5 12X RR) or sustained (LHN 12X RR) novel overexpression of c-myc was associated with RA resistance. RA-insensitive overexpression of MYCN by transduction in SMS-LHN also conferred RA resistance. Both parental and RA-resistant lines showed 2-4 logs of cell kill in response to N-(4-hydroxyphenyl)retinamide (4- HPR, fenretinide). Compared to parental lines, 4-HPR achieved 1-3 log greater cell kills in RA-resistant LHN 12X RR, LA-N-5 12X RR, KCNR 12X RR, and MYCN-transduced SMS-LHN or SK-N-RA. NB cell lines (n = 26) from 21 different patients showed that 16 of 26 (62%) were sensitive to 4-HPR (LC(90) < 10 microM), including lines established at relapse after myeloablative and/or 13-cis-RA therapy. CONCLUSION: Thus, RA-resistant NB cell lines can be sensitive (and in some cases collaterally hypersensitive) to 4-HPR.

Antineoplastic Agents↗

PubMed2000

Multidrug resistance-associated protein 1 (MRP1) expression in neuroblastoma cell lines and primary tumors.

BACKGROUND AND PROCEDURE: MRP1 expression by neuroblastomas was evaluated by Northern blot analysis in 21 cell lines and 90 primary untreated tumors. Cytotoxicity assay in cell lines was performed for five anticancer drugs used in treating neuroblastoma. RESULTS: MRP1 expression did not correlate with drug resistance or with MYCN RNA expression in cell lines. MRP1 expression was higher in drug-sensitive cell lines established after chemotherapy relative to cell lines at diagnosis, but highly drug-resistant cell lines showed low MRP1 expression. Positive expression of MRP1 RNA in primary tumors was associated with a poorer survival relative to MRP1-negative tumors. However, MRP1 expression levels did not correlate with age, stage, MYCN amplification, or MYCN expression, and higher MRP1 expression was not associated with a worse outcome. CONCLUSIONS: In neuroblastoma, positive MRP1 RNA expression at diagnosis has prognostic significance, but high drug resistance is conferred by mechanisms other than MRP1.

ATP-Binding Cassette Transporters↗

PubMed2000

Telomerase activity by TRAP assay and telomerase RNA (hTR) expression are predictive of outcome in neuroblastoma.

BACKGROUND: Recent studies have associated telomerase with prognostic factors and survival in neuroblastoma. PROCEDURE: We examined telomerase activity by telomere repeat amplification protocol (TRAP) and expression of the RNA component of telomerase (hTR) by Northern blotting in 106 primary neuroblastoma tumors and 22 established cell lines. RESULTS: Overall survival at 5 years for all 106 tumors was significantly better for patients with undetectable TRAP (75% vs. 59%; P = 0.03) or low hTR expression (84% vs. 43%; P < 0.0001), and especially for patients whose tumors had both low hTR expression and undetectable TRAP (all patients, 91% vs. 54%, P = 0.0002; for 17 stage IV-S tumors, 100% vs. 72%, P = 0.04). Strong expression of hTR was seen in 22 cell lines from aggressive tumors, and all maintained telomere length, but 3/22 were TRAP negative. CONCLUSIONS: These data suggest that both hTR expression and telomerase activity via the TRAP assay should be performed concurrently to predict survival in neuroblastoma patients, particularly in stage 4-S.

Child↗

PubMed2000

Synergism of buthionine sulfoximine and melphalan against neuroblastoma cell lines derived after disease progression.

BACKGROUND: Despite intensive-alkylator based regimens, >50% of patients with high-risk neuroblastoma (NB) die from recurrent disease that is probably due, in part, to acquired alkylator resistance. PROCEDURE: Using buthionine sulfoximine (BSO)-mediated, glutathione (GSH) depletion to modulate melphalan (L-PAM) resistance, we examined six NB cell lines established after progressive disease following either standard chemotherapy, BSO/L-PAM therapy, or myeloablative therapy and autologous hematopoietic stem cell transplant (AHSCT). RESULTS: Four of the six cell lines (three p53-nonfunctional and one p53-functional) showed high-level L-PAM resistance. CONCLUSIONS: Fixed ratio analysis demonstrated BSO/L-PAM synergy (combination index >1) for all cell lines tested. In L-PAM-resistant cell lines, the minimal cytotoxicity observed for BSO combined with nonmyeloablative concentrations of L-PAM was markedly enhanced (>4 logs total cell kill) when BSO was combined with myeloablative concentrations of L-PAM. In alkylator-resistant NB, the optimal use of BSO may require dose escalation of L-PAM to levels requiring AHSCT.

Antimetabolites, Antineoplastic↗

PubMed2000

Cross-resistance of topoisomerase I and II inhibitors in neuroblastoma cell lines.

PURPOSE: We have previously shown that neuroblastomal cell lines established from patients after intensive chemotherapy show sustained resistance to various drugs and especially high resistance to etoposide (up to 51 times higher than a clinically achievable level). To determine whether topoisomerase I inhibitors (topotecan and CPT- 11) are effective against etoposide-resistant neuroblastomas, we studied the response to topotecan and the active metabolite of CPT-11 (SN-38) in 19 cell lines with a spectrum of sensitivities to etoposide. MATERIALS AND METHODS: The panel included cell lines established at diagnosis and after disease progression either during induction chemotherapy or after myeloablative therapy supported with bone marrow transplantation. Cytotoxicities of topotecan, SN-38, and etoposide were determined using a microplate digital image microscopy (DIMSCAN) assay with a 4-log dynamic range. RESULTS: All six etoposide-resistant cell lines were resistant to topotecan and SN-38 (resistance defined as LC90 higher then clinically achievable levels for the drug). Significant cross-resistance by Pearson's correlation analysis (r > or = 0.6) occurred between topotecan + etoposide, topotecan + SN-38, and etoposide + SN-38. CONCLUSIONS: Topotecan and CPT-11 do not have significant activity against most etoposide-resistant neuroblastoma cell lines and this suggests that agents other than topoisomerase inhibitors should be explored for the treatment of recurrent neuroblastomas.

Antineoplastic Agents↗

PubMed2000

Neutrophils are cytotoxic and growth-inhibiting for neuroblastoma cells with an anti-GD2 antibody but, without cytotoxicity, can be growth-stimulating.

Neutrophils and mononuclear cells (MNC) can mediate antibody-dependent cellular cytotoxicity (ADCC) against cancer cells. To study cytotoxicity and growth inhibition of neuroblastoma cells by neutrophils and MNC with chimeric anti-disialoganglioside (GD2) monoclonal antibody (mAb) ch14.18, we developed digital image microscopy scanning (DIMSCAN) assays that measure fluorescence of target cells in 96-well plates after 6-18 h (cytotoxicity assay) or 7 days (growth assay). Neuroblastoma cell lines (GD2-positive: SMS-KCN, SMS-LHN, LA-N-1; GD2-negative: SK-N-SH) were preloaded with calcein acetoxymethyl ester for the cytotoxicity assay or labeled in situ after 7 days of culture with fluorescein diacetate in the growth assay. Fluorescence, as quantified by DIMSCAN, was correlated with neuroblastoma cell number in both assays (100-2000 cells/well). In the cytotoxicity test, both neutrophils and MNC effectively mediated ADCC of GD2-positive but not GD2-negative neuroblastoma cell lines. Cytotoxicity of both neutrophils and MNC increased with effector to target cell (E:T) ratio (5-50:1) and mAb ch.14.18 dose (0.1-10 microg/ml). ADCC of neutrophils, but not MNC, increased with addition of GM-CSF. Neutrophils, especially with rhGM-CSF, significantly suppressed growth of GD2-positive cell lines at a high E:T ratio (50:1) and mAb dose (10 microg/ml). Without antibody, neutrophils inhibited growth of one cell line (LA-N-1) but stimulated growth of two others (SMS-KCN, SMS-LHN). If neuroblastoma cells did not express GD2 (SK-N-SH), neutrophils stimulated growth whether or not antibody was present. Neutrophil culture supernatants increased growth of SK-N-SH, LA-N-1, and SMS-KCN cells, and MNC culture supernatants increased growth of SK-N-SH. In conclusion, neutrophils can mediate cytotoxicity and growth inhibition with a chimeric anti-GD2 antibody but also can promote tumor cell growth if antibody is not present or if GD2 is not expressed.

Antibodies, Monoclonal↗