Aerosol delivery of star polymer-siRNA nanoparticles as a therapeutic strategy to inhibit lung tumor growth.

Lung cancer is a major contributor to cancer-related death worldwide. siRNA nanomedicines are powerful tools for cancer therapeutics. However, there are challenges to overcome to increase siRNA delivery to solid tumors, including penetration of nanoparticles into a complex microenvironment following systemic delivery while avoiding rapid clearance by the reticuloendothelial system, and limited siRNA release from endosomes once inside the cell. Here we characterized cell uptake, intracellular trafficking, and gene silencing activity of miktoarm star polymer (PDMAEMA-POEGMA) nanoparticles (star nanoparticles) complexed to siRNA in lung cancer cells. We investigated the potential of nebulized star-siRNA nanoparticles to accumulate into orthotopic mouse lung tumors to inhibit expression of two genes [ßIII-tubulin, Polo-Like Kinase 1 (PLK1)] which: 1) are upregulated in lung cancer cells; 2) promote tumor growth; and 3) are difficult to inhibit using chemical drugs. Star-siRNA nanoparticles internalized into lung cancer cells and escaped the endo-lysosomal pathway to inhibit target gene expression in lung cancer cells in vitro. Nebulized star-siRNA nanoparticles accumulated into lungs and silenced the expression of ßIII-tubulin and PLK1 in mouse lung tumors, delaying aggressive tumor growth. These results demonstrate a proof-of-concept for aerosol delivery of star-siRNA nanoparticles as a novel therapeutic strategy to inhibit lung tumor growth.

Stathmin mediates neuroblastoma metastasis in a tubulin-independent manner via RhoA/ROCK signaling and enhanced transendothelial migration.

Neuroblastoma, the most common solid tumor of young children, frequently presents with aggressive metastatic disease and for these children the 5-year survival rates are dismal. Metastasis, the movement of cancer cells from one site to another, involves remodeling of the cytoskeleton including altered microtubule dynamics. The microtubule-destabilizing protein, stathmin, has recently been shown to mediate neuroblastoma metastasis although precise functions remain poorly defined. In this study we investigated stathmin's contribution to the metastatic process and potential mechanism(s) by which it exerts these effects. Stathmin suppression significantly reduced neuroblastoma cell invasion of 3D tumor spheroids into an extracellular matrix. Moreover, inhibiting stathmin expression significantly reduced transendothelial migration in two different neuroblastoma cell lines in vitro. Inhibition of ROCK, a key regulator of cell migration, in neuroblastoma cells highlighted that stathmin regulates transendothelial migration through ROCK signaling. Reduced stathmin expression in neuroblastoma cells significantly increased the activation of the RhoA small GTPase. Notably, re-expression of either wild type or a phospho-mimetic stathmin mutant (4E) made defective in tubulin binding returned cell migration and transendothelial migration back to control levels, indicating that stathmin may influence these processes in neuroblastoma cells independent of tubulin binding. Finally, stathmin suppression in neuroblastoma cells significantly reduced whole body, lung, kidney and liver metastases in an experimental metastases mouse model. In conclusion, stathmin suppression interferes with the metastatic process via RhoA/ROCK signaling in neuroblastoma cells. These findings highlight the importance of stathmin to the metastatic process and its potential as a therapeutic target for the treatment of neuroblastoma.

Delineating the Role of ßIV-Tubulins in Pancreatic Cancer: ßIVb-Tubulin Inhibition Sensitizes Pancreatic Cancer Cells to Vinca Alkaloids.

Pancreatic cancer (PC) is a lethal disease which is characterized by chemoresistance. Components of the cell cytoskeleton are therapeutic targets in cancer. ßIV-tubulin is one such component that has two isotypes-ßIVa and ßIVb. ßIVa and ßIVb isotypes only differ in two amino acids at their C-terminus. Studies have implicated ßIVa-tubulin or ßIVb-tubulin expression with chemoresistance in prostate, breast, ovarian and lung cancer. However, no studies have examined the role of ßIV-tubulin in PC or attempted to identify isotype specific roles in regulating cancer cell growth and chemosensitivity. We aimed to determine the role of ßIVa- or ßIVb-tubulin on PC growth and chemosensitivity. PC cells (MiaPaCa-2, HPAF-II, AsPC1) were treated with siRNA (control, ßIVa-tubulin or ßIVb-tubulin). The ability of PC cells to form colonies in the presence or absence of chemotherapy was measured by clonogenic assays. Inhibition of ßIVa-tubulin in PC cells had no effect chemosensitivity. In contrast, inhibition of ßIVb-tubulin in PC cells sensitized to vinca alkaloids (Vincristine, Vinorelbine and Vinblastine), which was accompanied by increased apoptosis and enhanced cell cycle arrest. We show for the first time that ßIVb-tubulin, but not ßIVa-tubulin, plays a role in regulating vinca alkaloid chemosensitivity in PC cells. The results from this study suggest ßIVb-tubulin may be a novel therapeutic target and predictor of vinca alkaloid sensitivity for PC and warrants further investigation.

Movers and shakers: cell cytoskeleton in cancer metastasis.

UNLABELLED: Metastasis is responsible for the greatest number of cancer deaths. Metastatic disease, or the movement of cancer cells from one site to another, is a complex process requiring dramatic remodelling of the cell cytoskeleton. The various components of the cytoskeleton, actin (microfilaments), microtubules (MTs) and intermediate filaments, are highly integrated and their functions are well orchestrated in normal cells. In contrast, mutations and abnormal expression of cytoskeletal and cytoskeletal-associated proteins play an important role in the ability of cancer cells to resist chemotherapy and metastasize. Studies on the role of actin and its interacting partners have highlighted key signalling pathways, such as the Rho GTPases, and downstream effector proteins that, through the cytoskeleton, mediate tumour cell migration, invasion and metastasis. An emerging role for MTs in tumour cell metastasis is being unravelled and there is increasing interest in the crosstalk between key MT interacting proteins and the actin cytoskeleton, which may provide novel treatment avenues for metastatic disease. Improved understanding of how the cytoskeleton and its interacting partners influence tumour cell migration and metastasis has led to the development of novel therapeutics against aggressive and metastatic disease. LINKED ARTICLES: This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24.

RNAi-mediated stathmin suppression reduces lung metastasis in an orthotopic neuroblastoma mouse model.

Metastatic neuroblastoma is an aggressive childhood cancer of neural crest origin. Stathmin, a microtubule destabilizing protein, is highly expressed in neuroblastoma although its functional role in this malignancy has not been addressed. Herein, we investigate stathmin's contribution to neuroblastoma tumor growth and metastasis. Small interfering RNA (siRNA)-mediated stathmin suppression in two independent neuroblastoma cell lines, BE(2)-C and SH-SY5Y, did not markedly influence cell proliferation, viability or anchorage-independent growth. In contrast, stathmin suppression significantly reduced cell migration and invasion in both the neuroblastoma cell lines. Stathmin suppression altered neuroblastoma cell morphology and this was associated with changes in the cytoskeleton, including increased tubulin polymer levels. Stathmin suppression also modulated phosphorylation of the actin-regulatory proteins, cofilin and myosin light chain (MLC). Treatment of stathmin-suppressed neuroblastoma cells with the ROCKI and ROCKII inhibitor, Y-27632, ablated MLC phosphorylation and returned the level of cofilin phosphorylation and cell invasion back to that of untreated control cells. ROCKII inhibition (H-1152) and siRNA suppression also reduced cofilin phosphorylation in stathmin-suppressed cells, indicating that ROCKII mediates stathmin's regulation of cofilin phosphorylation. This data demonstrates a link between stathmin and the regulation of cofilin and MLC phosphorylation via ROCK. To examine stathmin's role in neuroblastoma metastasis, stathmin short hairpin RNA (shRNA)\luciferase-expressing neuroblastoma cells were injected orthotopically into severe combined immunodeficiency-Beige mice, and tumor growth monitored by bioluminescent imaging. Stathmin suppression did not influence neuroblastoma cell engraftment or tumor growth. In contrast, stathmin suppression significantly reduced neuroblastoma lung metastases by 71% (P<0.008) compared with control. This is the first study to confirm a role for stathmin in hematogenous spread using a clinically relevant orthotopic cancer model, and has identified stathmin as an important contributor of cell invasion and metastasis in neuroblastoma.

ß-blockers increase response to chemotherapy via direct antitumour and anti-angiogenic mechanisms in neuroblastoma.

BACKGROUND: The use of ß-blockers for the management of hypertension has been recently associated with significant clinical benefits in cancer patients. Herein, we investigated whether ß-blockers could be used in combination with chemotherapy for the treatment of neuroblastoma. METHODS: Seven ß-blockers were tested for their antiproliferative and anti-angiogenic properties alone, and in combination with chemotherapy in vitro; the most potent drug combinations were evaluated in vivo in the TH-MYCN mouse model of neuroblastoma. RESULTS: Three ß-blockers (i.e., carvedilol, nebivolol and propranolol) exhibited potent anticancer properties in vitro and interacted synergistically with vincristine, independently of P-glycoprotein expression. ß-blockers potentiated the anti-angiogenic, antimitochondrial, antimitotic and ultimately pro-apoptotic effects of vincristine. In vivo, ß-blockers alone transiently slowed tumour growth as compared with vehicle only (P<0.01). More importantly, when used in combination, ß-blockers significantly increased the tumour regression induced by vincristine (P<0.05). This effect was associated with an increase in tumour angiogenesis inhibition (P<0.001) and ultimately resulted in a four-fold increase in median survival, as compared with vincristine alone (P<0.01). CONCLUSION: ß-blockers can increase treatment efficacy against neuroblastoma, and their combination with chemotherapy may prove beneficial for the treatment of this disease and other drug-refractory cancers.

Endothelial cell dysfunction and cytoskeletal changes associated with repression of p16(INK4a) during immortalization.

The immortalization process is a fundamental step in the development of most (if not all) human cancers, including the aggressive endothelial cell (EC)-derived malignancy angiosarcoma. Inactivation of the tumor suppressor p16(INK4a) and the development of multiple chromosomal abnormalities are features of angiosarcoma that are recapitulated during telomerase-mediated immortalization of human ECs in vitro. The present study used a panel of telomerase-immortalized bone marrow EC (BMEC) lines to define the consequences of inactivation of p16(INK4a) on EC function and to identify molecular changes associated with repression of p16(INK4a). In a comparison of two immortalized BMEC mass cultures and six clones, the cell lines that repressed p16(INK4a) showed a higher rate of proliferation and an impaired ability to undergo morphogenic differentiation and form vessel-like structures in vitro. Proteomic comparison of a p16(INK4a)-negative and a p16(INK4a)-positive BMEC mass culture at early- and late-passage time points following transduction with telomerase reverse transcriptase (hTERT) revealed altered expression of cytoskeletal proteins, including vimentin and α-tropomyosin (αTm), in the immortal cells. Immunoblot analyses of a panel of 11 immortal clones showed that cells that lacked p16(INK4a) expression tended to accumulate more dramatic changes in these cytoskeletal proteins than cells that retained p16(INK4a) expression. This corresponded with aberrant cytoskeletal architectures among p16(INK4a)-negative clones, which featured thicker actin stress fibers and less fluid membrane ruffles than p16(INK4a)-positive cells. A direct link between p16(INK4a) repression and defective EC function was confirmed by analysis of normal cells transfected with small interfering RNA (siRNA) targeting p16(INK4a). siRNA-mediated repression of p16(INK4a) significantly impaired random motility and vessel formation in vitro. This report is the first to demonstrate that ECs that repress the expression of p16(INK4a) are prone to defects in motility, morphogenesis and cytoskeletal organization. These defects are likely to reflect alterations that occur during the development of EC-derived malignancies.

TRIM16 acts as a tumour suppressor by inhibitory effects on cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells.

The family of tripartite-motif (TRIM) proteins are involved in diverse cellular processes, but are often characterized by critical protein-protein interactions necessary for their function. TRIM16 is induced in different cancer types, when the cancer cell is forced to proceed down a differentiation pathway. We have identified TRIM16 as a DNA-binding protein with histone acetylase activity, which is required for the retinoic acid receptor ß(2) transcriptional response in retinoid-treated cancer cells. In this study, we show that overexpressed TRIM16 reduced neuroblastoma cell growth, enhanced retinoid-induced differentiation and reduced tumourigenicity in vivo. TRIM16 was only expressed in the differentiated ganglion cell component of primary human neuroblastoma tumour tissues. TRIM16 bound directly to cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells. TRIM16 reduced cell motility and this required downregulation of vimentin. Retinoid treatment and enforced overexpression caused TRIM16 to translocate to the nucleus, and bind to and downregulate nuclear E2F1, required for cell replication. This study, for the first time, demonstrates that TRIM16 acts as a tumour suppressor, affecting neuritic differentiation, cell migration and replication through interactions with cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells.

LIM-kinase 2, a regulator of actin dynamics, is involved in mitotic spindle integrity and sensitivity to microtubule-destabilizing drugs.

LIM-kinase 2 (LIMK2) belongs to the LIMK family of proteins, which comprises LIMK1 and LIMK2. Both proteins regulate actin polymerization through phosphorylation and inactivation of the actin depolymerizing factor cofilin. In this study, we show that the level of LIMK2 protein is increased in neuroblastoma, BE(2)-C cells, selected for resistance to microtubule-destabilizing agents, vincristine and colchicine. However, the level of phosphorylated LIMK1 and LIMK2 was similar in the resistant and parental BE(2)-C cells. In contrast, the level of phospho-cofilin was greatly increased in the drug-resistant cells. Downregulation of LIMK2 expression increases sensitivity of neuroblastoma SH-EP cells to vincristine and vinblastine but not to microtubule-stabilizing agents, while it's overexpression increased its resistance to vincristine. Its vincristine-induced mitotic arrest was moderately inhibited in the LIMK2 knockdown cells, suggesting that the increased drug sensitivity is through an alternative mechanism other then mitotic arrest and apoptosis. Moreover, downregulation of LIMK2 expression induces formation of abnormal mitotic spindles, an effect enhanced in the presence of microtubule-destabilizing agents. LIMK2 is important for normal mitotic spindle formation and altered LIMK2 expression mediates sensitivity to microtubule destabilizing agents. These findings suggest that inhibition of LIMK2 activity may be used for the treatment of tumors resistant to microtubule-destabilizing drugs.

Improving the targeting of tubulin-binding agents: lessons from drug resistance studies.

Natural product drugs that target the tubulin/microtubule system remain an important component in the therapeutic arsenal to treat many types of malignancies. Agents such as the taxanes and vinca alkaloids bind to beta-tubulin and disrupt microtubule dynamics by inducing a potent mitotic block and subsequent cell death. Understanding why certain cancers do not respond to treatment or develop resistance has been the subject of numerous studies in recent years. An increasing body of evidence suggests that alterations in the drug target, such as tubulin mutations, altered microtubule dynamics, altered tubulin isotype expression, and modifications in microtubule regulatory proteins, are key mechanisms of antimicrotubule drug resistance. In addition, recent work indicates that other cytoskeletal proteins that can regulate microtubule dynamics through signaling or structural interactions may be important determinants of antimicrotubule resistance. As our understanding of drug action and resistance mechanisms has increased, we can now begin to exploit these to design strategies that overcome, or counteract resistance, hence improving the efficacy of antimicrotubule agents for the treatment of cancer. This review highlights the major areas of investigation as they relate to the tubulin/microtubule system and discusses opportunities that potentially exist for improved therapeutic benefit in the treatment of drug resistant disease.

Markedly diminished drug resistance-inducing properties of vinflunine (20',20'-difluoro-3',4'-dihydrovinorelbine) relative to vinorelbine, identified in murine and human tumour cells in vivo and in vitro.

PURPOSE: Vinflunine (VFL) is a novel Vinca alkaloid with markedly superior experimental in vivo antitumour activity to its parent molecule, vinorelbine (Navelbine, NVB), against a panel of murine and human tumours. The aim of this study was to establish whether there are differences in the rate and extent of development of resistance, both in vivo and in vitro, to these two newer Vinca alkaloids under identical selection conditions. METHODS: Using P388 leukaemia cells in vivo, it was evident that VFL induced drug resistance far less readily than NVB, as shown by the number of passages required to select for total resistance. Under in vitro conditions, using A549 human lung carcinoma cells, it was also clearly shown by drug sensitivity determinations that VFL was a less-potent inducer of drug resistance than NVB. Resistance resulting from either in vivo or in vitro selection was associated with a classic multidrug resistance profile. Further characterization of the drug-resistance phenotype of the most highly resistant A549 sublines showed that the level of total beta-tubulin expression appeared to be modified exclusively in the NVB-resistant cells. CONCLUSION: The clear demonstration that resistance to VFL developed far less readily than resistance to NVB both in vivo and in vitro may have potential clinical implications.

Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells.

Vinca alkaloids are used extensively in the treatment of childhood acute lymphoblastic leukemia (ALL) and despite their usefulness, drug resistance remains a serious clinical problem. Vinca alkaloids bind to the beta-tubulin subunit of the alpha/beta-tubulin heterodimer and inhibit polymerization of microtubules. Recent studies have implicated altered beta-tubulin isotype expression and mutations in resistance to microtubule-stabilizing agents. Microtubule-associated protein (MAP) MAP4 binds to and stabilizes microtubules, and increased expression is associated with decreased sensitivity to microtubule-depolymerizing agents. To address the significance of beta-tubulin and MAP4 alterations in childhood ALL, two CCRF-CEM-derived Vinca alkaloid resistant cell lines, VCR R (vincristine) and VLB100 (vinblastine), were examined. Decreased expression of class III beta-tubulin was detected in both VCR R and VLB100 cells. VCR R cells and to a lesser extent VLB100 cells expressed increased levels of MAP4 protein. Increased microtubule stability was observed in these VCR R cells as identified by the high levels of polymerized tubulin (45.6 +/- 2.6%; P < 0.005) compared with CEM and VLB100 cells (24.7 +/- 3.3% and 24.7 +/- 2.5%, respectively). Expression was associated with a single MAP4 isoform in the polymerized microtubule fraction in CEM and VCR cells. In contrast, VLB100 cells expressed a lower molecular weight isoform in the polymerized fraction. Two-dimensional-PAGE and immunoblotting revealed marked posttranslational changes in class I beta-tubulin in VCR R cells not evident in CEM cells. Sequencing of the beta-tubulin (HM40) gene identified a point mutation in VCR R cells in nucleotide 843 (CTC-->ATC; Leu(240)-->Ile) that was not present in CEM or VLB100 cells. This mutation resides in a region of beta-tubulin that lies in close proximity to the alpha/beta tubulin interface. Multiple alterations related to normal microtubule function were identified in ALL cells selected for resistance to Vinca alkaloids, and these alterations may provide important insight into mechanisms mediating resistance to Vinca alkaloids.

Anticancer therapy with novel tubulin-interacting drugs.

Antimitotic agents that target tubulin, including the taxanes and vinca alkaloids, are important components of current anticancer therapy. Whilst these antimitotic drugs are highly effective in the treatment of a number of cancers, both acquired and intrinsic resistance to these agents is a major clinical problem. Furthermore, the systemic toxicity, and in some cases lack of oral availability, make these agents less than ideal. Recently much effort has been directed on the isolation and synthesis of new antimitotic drugs that target the tubulin/microtubule system and display efficacy against drug-refractory carcinomas. Newly described compounds include structurally diverse natural products, such as dolastatin, epothilones and discodermolide, derivatives and structural analogues of traditional antimitotics, and novel synthetic molecules. Additionally, new developments in drug targeting are improving efficacy and therapeutic indices of traditional agents. A number of promising 'new generation' antimitotics are now undergoing clinical testing. These new agents are reviewed here in terms of their mechanism(s) of action on microtubules, effectiveness against drug-resistant tumour cells and clinical potential.

Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells.

The pro-apoptotic protein, Bax, has been reported to translocate from cytosol to mitochondria following exposure of cells to apoptotic stresses including cytokine withdrawal and treatment with glucocorticoids and cytotoxic drugs. These observations, coupled with reports showing that Bax causes the release of mitochondrial cytochrome c, implicate Bax as a central mediator of the apoptotic process. In this report we demonstrate by subcellular fractionation a significant shift in Bax localization from cytosol to cellular membranes in two human tumor cell lines exposed to staurosporine or etoposide. Immunofluorescence studies confirmed that Bax specifically relocalized to the mitochondria. This redistribution of Bax occurred in concert with, or just prior to, proteolytic processing of procaspase-3, activation of DEVD-specific cleavage activity and degradation of poly(ADP-ribose) polymerase. However, Bax membrane translocation was independent of caspase activity as determined using the broad-range caspase inhibitor z-VAD-fmk. High level overexpression of the anti-apoptotic protein Bcl-2 prevented Bax redistribution to the mitochondria, caspase activation and apoptosis following exposure to staurosporine or etoposide. These data confirm the role of Bax in mitochondrial cytochrome c release, and indicate that prevention of Bax translocation to the mitochondrial membrane represents a novel mechanism by which Bcl-2 inhibits drug-induced apoptosis.

Antisense oligonucleotides to class III beta-tubulin sensitize drug-resistant cells to Taxol.

A major impediment to the successful use of Taxol in the treatment of cancer is the development of drug resistance. The major cellular target of Taxol is the microtubule that is comprised of alpha- and beta-tubulin heterodimers. Binding sites for Taxol have been delineated on the beta-tubulin subunit that has six isotypes. We have recently described increased expression of the brain-specific human class III beta-tubulin isotype, encoded by the Hbeta4 gene, in both Taxol-resistant ovarian tumours and non-small-cell lung cancer cell lines. To evaluate directly the role of the class III beta-tubulin isotype in mediating Taxol resistance, antisense phosphorothioate oligodeoxynucleotides (ODN) targeted against various regions of the Hbeta4 gene have been designed and examined for their efficacy in reducing Hbeta4 gene and protein expression. Taxol-resistant lung cancer cells, A549-T24, which are 17-fold resistant to Taxol and display a fourfold increase in Hbeta4 expression compared to the parental A549 cells, were treated with 1 microM antisense ODNs. Two ODNs, AS1 and AS3, were found to reduce mRNA expression by 40-50%, as determined by reverse transcription polymerase chain reaction. A concentration-dependent reduction in Hbeta4 mRNA expression was demonstrated with AS1 ODN. Immunofluorescence staining of cells treated with AS1 ODN revealed a decrease in class III protein expression which corresponded to a 39% increase in sensitivity to Taxol (P < 0.005). These findings support an important role for Hbeta4 (class III) beta-tubulin expression in Taxol resistance and have potential implications for the treatment of Taxol-resistant tumours.

Altered expression of the MYCN oncogene modulates MRP gene expression and response to cytotoxic drugs in neuroblastoma cells.

We have recently shown a close correlation between expression of the Multidrug Resistance-associated Protein (MRP) gene and the MYCN oncogene and provided evidence that high MRP expression is a powerful independent predictor of poor outcome in neuroblastoma (Norris et al., New Engl. J. Med., 334, 231-238, 1996). The effect of MYCN down-regulation on MRP expression and response to cytotoxic drugs was investigated in NBL-S neuroblastoma cells transfected with MYCN antisense RNA constructs. Concomitant with MYCN down-regulation, the level of MRP expression was decreased in the NBAS-4 and NBAS-5 antisense transfectants. These cells demonstrated significantly increased sensitivity to the high affinity MRP substrates vincristine, doxorubicin, sodium arsenate and potassium antimony tartrate, but not to the poor MRP substrates, taxol or cisplatin. Similarly, transfection of full-length MYCN cDNA into SH-EP neuroblastoma cells resulted in increased MRP expression and significantly increased resistance specifically to MRP substrates. The results provide evidence for the MYCN oncogene influencing cytotoxic drug response via regulation of MRP gene expression. Our data also provide a link between the malignant and chemoresistant phenotypes of this childhood malignancy.

P-glycoprotein-mediated methotrexate resistance in CCRF-CEM sublines deficient in methotrexate accumulation due to a point mutation in the reduced folate carrier gene.

We have previously described a series of methotrexate (MTX)-selected CCRF-CEM sublines (CEM/MTX R1-3) displaying increased resistance to drugs associated with the multidrug resistance phenotype and have provided evidence that MDR1 P-glycoprotein contributes to multifactorial MTX resistance in these cells. We have also suggested that P-glycoprotein-mediated MTX transport arises in these cells due to a deficiency in the normal MTX transport route, the reduced folate carrier (RFC). We have now determined the nucleotide sequence of the RFC gene in CEM/MTX R1-3 cells and confirm that the carrier is defective in these cells as a result of a premature stop mutation at codon 99, which severely truncates the encoded protein. CEM/MTX R3 cells were removed from MTX, and a series of sublines with increasing MDR1 expression were derived, following selection with vincristine. These cells show increasing cross-resistance to vincristine as well as other drugs associated with the multidrug resistance phenotype. More importantly, the increased P-glycoprotein expression correlates with increased resistance to MTX, supporting the hypothesis that in cells with a defective carrier protein, MTX can become a substrate for P-glycoprotein. Our data have implications for the P-glycoprotein-mediated transport of other hydrophilic drugs in situations where the relevant carrier protein has been functionally inhibited.

In vitro effects of MYCN sense and antisense expression in MYCN-amplified human neuroblastoma cells.

Amplification of the MYCN oncogene is a strong predictor of treatment failure and chemo-resistance in childhood neuroblastoma. Stable expression of two partial MYCN gene fragments in antisense orientation reduced Mycn protein expression in an MYCN-amplified neuroblastoma tumor cell line, however, antisense cells did not exhibit an increased in vitro sensitivity to cytotoxic or differentiating agents. In contrast, partial MYCN sense transfectants exhibited increased resistance to cytotoxic drugs. These data suggest that the chemo-resistance of MYCN-amplified neuroblastoma cells is complex, and may be due to factors additional to Mycn protein expression.