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Biomedical subjects

V Ling

Publications and source records attributed to V Ling.

At least 55 records · Page 3Linked to original sources
PubMed1997

Extraction of Hoechst 33342 from the cytoplasmic leaflet of the plasma membrane by P-glycoprotein.

P-glycoprotein is an ATP-dependent plasma membrane multidrug transporter of broad specificity. A common chemical property of its substrates is that all are lipophilic. Using Hoechst 33342 as the substrate, we have previously shown that P-glycoprotein extracts the substrate directly from the lipid bilayer [Shapiro, A. B., Corder, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 115-121]. In this paper, we determined the leaflet of the plasma membrane from which P-glycoprotein extracts Hoechst 33342. The initial rate of Hoechst 33342 transport upon ATP addition to P-glycoprotein-rich inside-out plasma membrane vesicles decreased slightly with the amount of time previously elapsed for slow diffusion of Hoechst 33342 to the extracellular leaflet. This result is consistent with transport from the cytoplasmic leaflet. Fluorescence resonance energy transfer from donor Hoechst 33342 to acceptor 2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-sn-glycero- 3-phosphocholine (Nbd-C6-HPC) in the cytoplasmic leaflet was used to monitor the amount of Hoechst 33342 in the cytoplasmic leaflet versus time. The initial rate of decrease of the energy-transfer-related Nbd-C6-HPC fluorescence after ATP addition exceeded that of the Hoechst 33342 fluorescence and continued to decrease after decrease of the Hoechst 33342 fluorescence had ceased. These effects were consistent with transport of Hoechst 33342 from the cytoplasmic leaflet to the aqueous interior of the vesicles, followed by rebinding to the extracellular leaflet. This demonstrates that P-glycoprotein transports drugs from the cytoplasmic leaflet of the plasma membrane directly to the aqueous extracellular medium. This finding has implications for efforts to localize the drug-binding site(s) within P-glycoprotein.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

Positively cooperative sites for drug transport by P-glycoprotein with distinct drug specificities.

In this paper, we show that P-glycoprotein contains two distinct sites for drug binding and transport, and that, unexpectedly, these sites interact in a positively cooperative manner. The kinetics of transport of rhodamine 123 and Hoechst 33342 in isolated P-glycoprotein-rich plasma membrane vesicles from Chinese hamster ovary CH(R)B30 cells were followed by continuous fluorescence monitoring. Each substrate stimulated P-glycoprotein-mediated transport of the other. Colchicine and quercetin stimulated rhodamine 123 transport and inhibited Hoechst 33342 transport. In contrast, anthracyclines such as daunorubicin and doxorubicin stimulated Hoechst 33342 transport and inhibited rhodamine 123 transport. Vinblastine, actinomycin D, and etoposide inhibited transport of both dyes. The results are consistent with a functional model of P-glycoprotein containing at least two positively cooperative sites (H site and R site) for drug binding and transport. This model is consistent with earlier observations of competitive and non-competitive effects of P-glycoprotein substrates and chemosensitizers. Such a two-site model may be fundamental to multidrug transport by P-glycoprotein, and it may be a feature common to other ATP-dependent transporters belonging to the ATP-binding cassette superfamily.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Formulation development and primary degradation pathways for recombinant human nerve growth factor.

The chemical and physical stabilities of recombinant human nerve growth factor (NGF) in aqueous solution were investigated between 5 and 37 degrees C and at pH 4.2-5.8. NGF chemical stability decreased with a decrease in pH due to Asp60-Pro61 cleavage, with the stability being greater in acetate buffer than in succinate buffer at each pH investigated. Aggregation was a significant degradation pathway at 37 degrees C, with the aggregation rate being greatest in succinate buffer at pH 5.8. Quantitation of NGF degradation by cation-exchange chromatography was complicated by the rearrangement of the NGF monomer variants into various mixed dimers over time. Treatment with dilute acid brought the dimer distribution rapidly to equilibrium, allowing NGF degradation to be accurately quantitated. An acetate-buffered formulation at pH 5.5 was investigated in more detail. To assist in degradation product identification, NGF degradation was accelerated with base, hydrogen peroxide, and temperature. These degradation products were shown to coelute on RP-HPLC with the variants found when the protein was stored at -70, 5, and 25 degrees C. By electrospray mass spectrometry, peptide maps, and LC/MS, these degradation products were shown to be monooxidized (Met37) and dioxidized (Met37 and Met92) NGF, with Met37 being more labile, deamidated NGF (Asn45), and NGF with Asp93 isomerized to beta-Asp93. NGF can be stored in pH 5.5 acetate buffer at 5 degrees C for 1.5 years with less than 10% conversion to these degradation products, with Asp93 isomerization being the primary degradation pathway.

Amino Acid Sequence↗
PubMed1997

Sensitive immunofluorescence detection of the expression of P-glycoprotein in malignant cells.

Because reversal of multidrug resistance increases chemotoxicity, early detection of low P-glycoprotein expression is clinically relevant for justifying early treatment of those patients that might benefit most from reversal therapy. We elected to score P-glycoprotein in single tumor cells, because the gene is rarely amplified, mRNA levels do not necessarily correlate with protein levels, and many normal hematopoietic or stroma cells within tumors and leukemic marrows also express P-glycoprotein. We enhanced the "signal-to-noise" ratio for detecting low P-glycoprotein levels by a novel complex made by pre-incubating mouse peroxidase-antiperoxidase, used solely to provide a stable framework for attaching multiple DTAF-labeled F(ab')2 fragments of rabbit antimouse IgG. We improved specificity by using both C219 and C494, which are directed against separate internal P-glycoprotein epitopes. We standardized staining with two series of negative and positive controls, in which P-glycoprotein was quantified by immunoblot, and confirmed sensitivity by staining a low-expression cell line and "mixed" samples containing small numbers of positive cells. We measured P-glycoprotein by flow cytometry, examining aliquots by differential interference contrast microscopy to identify malignant cells, in which we confirmed P-glycoprotein staining by fluorescence microscopy. We detected low P-glycoprotein expression in clinical samples of leukemic blasts, distinguishing them from normal P-glycoprotein-expressing hematopoietic cells. This assay may be valuable for early diagnosis of low, but potentially important expression of P-glycoprotein, thereby allowing early application of reversal therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Multidrug resistance protein (MRP) expression in retinoblastoma correlates with the rare failure of chemotherapy despite cyclosporine for reversal of P-glycoprotein.

Failure of chemotherapy associated with expression of the multidrug resistance protein p170 frequently occurs in retinoblastoma (RB). Despite using cyclosporine, which inhibits p170 and improves our chemotherapy results, rare failures occur. In nonmetastatic primarily enucleated RBs, we show expression of p170 in 3 of 18 samples and expression of multidrug resistance protein (MRP), the second protein associated with resistance to chemotherapy, in 1 of 18 samples. All three RBs that failed chemotherapy without cyclosporine expressed MRP with p170. All three RBs that were enucleated immediately when chemotherapy failed despite the addition of cyclosporine expressed only MRP. One RB enucleated 2 years after failing chemotherapy with cyclosporine, despite radiation and salvage chemotherapy, expressed both p170 and MRP. Two metastatic RBs that expressed both p170 and MRP at diagnosis and at recurrence failed chemotherapy without cyclosporine, whereas one metastatic RB that expressed neither protein was cured by chemotherapy without cyclosporine. MRP may result in failure of chemotherapy despite the elimination of p170-expressing clones by cyclosporine.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Isolation and characterization of daunorubicin-resistant AML-2 sublines.

An attempt was made to isolate resistant sublines of acute myelogenous leukemia (OCI/ AML-2) cells by chronic exposure to gradually increasing concentrations of daunorubicin in order to determine the mechanism of its resistance to this drug. Four daunorubicin-resistant sublines, AML-2/D100, /D250, /D500, and /D1,000 were isolated. The values of relative resistance of each daunorubicin-resistant AML subline were about 3, 6, 18, and 23-fold, respectively, as compared to the AML-2 line with an IC50 of 5 nM. The daunorubicin-resistant AML-2 sublines also showed cross resistance to various anticancer drugs including another anthracycline doxorubicin, a Vinca alkaloid vincristine, and an epipodophyllotoxin etoposide. A functional assay using flow cytometry showed decreased accumulation of daunorubicin in these sublines as compared to that of AML-2, which was reversed by cyclosporin A or cyanide. The development of the ATP-dependent multidrug resistant phenotype was due to low to high levels of expression of P-glycoprotein (PGP). The major mechanisms of increased PGP appears to be associated with gene amplification. In addition, other mechanisms such as increased stability of protein or mRNA might be involved depending on the concentration of daunorubicin used for selection. However, a multidrug resistance-associated protein (MRP) was not involved in these resistant sublines. These daunorubicin-resistant AML-2 sublines could provide a useful model for the study of multidrug resistance mediated by PGP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Effect of quercetin on Hoechst 33342 transport by purified and reconstituted P-glycoprotein.

Multidrug resistance due to P-glycoprotein is a serious impediment to successful chemotherapy of cancer. Numerous compounds are known that inhibit the drug-exporting function of P-glycoprotein. Understanding the mechanisms of action of these chemosensitizers is made difficult by the complexity of the in vivo cell systems usually employed. To examine the direct effects of chemosensitizers, we have developed a system in which purified and reconstituted P-glycoprotein transports. Hoechst 33342 from the lipid membrane to the aqueous interior of proteoliposomes, requiring ATP hydrolysis (Shapiro AB and Ling V, J Biol Chem 270: 16167-16175, 1995). Here, we use this system to understand the effect on P-glycoprotein of quercetin, one of three flavonoids that have been reported to have the unique property of stimulating drug transport by P-glycoprotein in vivo (Phang et al., Cancer Res 53: 5977-5981, 1993). Since flavonoids are abundant in food, it is important to understand their effects on the function of P-glycoprotein because of the implications for cancer chemotherapy. In our hands, quercetin inhibited P-glycoprotein-mediated Hoechst 33342 efflux and enhanced accumulation, as measured by flow cytometry, by multidrug-resistant CHRC5 cells. In the purified system, quercetin strongly inhibited Hoechst 33342 transport by P-glycoprotein, at least in part by inhibiting the ATPase activity of P-glycoprotein required for transport. We conclude that the previously reported stimulatory effect of quercetin on drug efflux from multidrug-resistant cells is not a direct effect on P-glycoprotein. The ATPase domain of P-glycoprotein may be an attractive target for new chemosensitizing agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

In vitro differentiation of embryonic stem cells: immunophenotypic analysis of cultured embryoid bodies.

The process of in vitro embryonic stem cell differentiation and embryoid body development was monitored using a panel of antibodies against surface markers traditionally associated with embryonic tissue (Forssman, SSEA-1) and hematopoietic progenitor cells (Fall-3, HSA, Sca-1, Thy-1.2, ER-MP12, CD45, AA4.1, and c-kit). All markers with the exception of CD45 and AA4.1 were initially detected in cultures of undifferentiated ES cells. During the first 11 days of differentiation, distinct and reproducible patterns of surface expression were observed for each marker. Using the kinetic display of surface markers as a gauge of differentiation, perturbations in embryoid body development were detected in cultures supplemented with interleukin-11, a gp130-activating cytokine thought to affect embryonic stem cell differentiation. In the absence of exogenous cytokines, microbead immunoselected day 7 c-kit, ER-MP12, and CD45-positive embryoid body cells were enriched for hematopoietic progenitors as detected by methylcellulose colony assays, while no significant enrichment of hematopoietic progenitors was observed with Sca-1, Thy-1.2, Fall-3, and Forssman-immunoselected cells. These results indicate that the process of early embryoid body development is associated with a programmed sequence of cell surface marker display, concomitant with the development of phenotypically definable embryonic cell lineages.

Animals↗
PubMed1997

Structural identification of the hematopoietic progenitor antigen ER-MP12 as the vascular endothelial adhesion molecule PECAM-1 (CD31).

The monoclonal antibody ER-MP12 was recently described to recognize an antigen present on cell subpopulations of adult mouse bone marrow, including pluripotent hematopoietic stem cells. In an effort to understand the function of ER-MP12 antigen in hematopoiesis, we used biochemical and physical methods to determine its identity. ER-MP12 antigen was isolated by immunoprecipitation from FDCP-1 cell membrane proteins, yielding a glycosylated 113-kDa band upon analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thirteen peptides derived from trypsinized ER-MP12 antigen were analyzed by electrospray ionization mass spectrometry and compared to a protein sequence database. The search revealed the identity of the ER-MP12 antigen as platelet endothelial cell adhesion molecule-1, CD31 (PECAM-1). This result was subsequently confirmed by Edman sequencing of a single ER-MP12 peptide fragment followed by comparison with PECAM-1 sequence. In addition, flow cytometric analysis of bone marrow and embryonic stem cells revealed highly similar profiles between ER-MP12 and CD31 (MEC 13.3 antibody)-stained cells. The presence of PECAM-1 on primitive hematopoietic stem cells supports the theory for the interaction of hematopoietic progenitor and stem cells with bone marrow stroma and transendothelial migration.

Amino Acid Sequence↗
PubMed1997

Multidrug resistance: molecular mechanisms and clinical relevance.

Multidrug resistance (MDR) describes the phenomenon of simultaneous resistance to unrelated drugs. It has been a decade since the P-glycoprotein (Pgp) gene, which is associated with a form of MDR caused by reduced drug accumulation, was cloned. Thus, this would seem to be an appropriate time to evaluate our understanding of this form of MDR. The two MDR genes identified in humans to date (the MDR-associated protein [MRP] and Pgp genes) are structurally similar and both are members of the ATP-binding cassette (ABC) transporter family. Although the physiological role of MRP is not yet understood, one Pgp gene (mdr1) plays an important role in the blood-tissue barrier and the other (mdr2/3) is involved in phospholipid transport in the liver. A variety of compounds (chemosensitizing agents) can interfere with Pgp and MRP function; such agents may improve the efficacy of conventional therapy when used in combination with such regimens. Determining the roles cellular MDR mechanisms play in patients' response to chemotherapy is a major challenge. Using Pgp and MRP as molecular markers to detect MDR tumor cells is technically demanding, and solid tumors in particular contain heterogeneous cell populations. Since MDR requires Pgp or MRP gene expression, clinically relevant gene expression thresholds need to be established; sequential samples from individual patients are valuable for correlating MDR gene expression with the clinical course of disease. Studies in leukemias, myelomas, and some childhood cancers show that Pgp expression correlates with poor response to chemotherapy. However, in some cases, inclusion of a reversing or chemosensitizing agent such as verapamil or cyclosporin A has improved clinical efficacy. Such agents may inactivate Pgp in tumor cells or affect Pgp function in normal cells, resulting in altered pharmacokinetics. It would be interesting to determine whether patients who fail treatment in the presence of chemosensitizing agents acquire other MDR mechanisms. The ABC transporter superfamily in prokaryotes and eukaryotes is involved in the transport of substrates ranging from ions to large proteins. Of the 15 or more ABC transporter genes characterized in human cells, two (Pgp and MRP) cause MDR. Therefore, it would be relevant to determine the number of such genes present in the human genome; however, extrapolating from the number of ABC transporter genes in bacteria, the human gene probably contains a minimum of 200 ABC transporter superfamily members. Thus, tumor cells can potentially use many ABC transporters to mount resistance to known and future therapeutic agents. The challenge will be to determine which ABC transporters are clinically relevant. Despite the potential of tumor cells to protect themselves, a variety of malignancies can be successfully treated with chemotherapy. This may provide unique insights.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

A novel energy dependent mechanism reducing daunorubicin accumulation in acute myeloid leukemia.

Using cyclosporin A (CsA) to inhibit P-glycoprotein (P-gp) function we showed previously that there was a discordance between the ability of acute myeloid leukemic (AML) blast cells to accumulate daunorubicin and P-gp antigen expression (Xie et al, Leukemia 1995; 9:1882-1887). This discordance suggests that a CsA-sensitive drug efflux mechanism distinct from P-gp is expressed in many clinical samples. In the present study using the ATP depleting agents cyanide, azide, or dinitrophenol to inhibit energy dependent transport processes, we observed even larger increases in daunorubicin accumulation than were seen with CsA. Similar patterns were seen in a wide range of P-gp negative human cancer cell lines. Also the observed cyanide effect did not correlate with the expression of mRNA for multidrug resistance-associated protein (MRP), the only other member of the ABC family of membrane transporters that is known to be capable of effluxing daunorubicin. Thse results suggest that daunorubicin accumulation in many cases of AML is modulated by one or more novel energy-dependent processes that are distinct from P-gp or MRP. We speculate that this novel drug transport mechanism(s) may influence the response of AML patients to daunorubicin and other therapeutic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Paroxysmal nocturnal hemoglobinuria: new insights from murine Pig-a-deficient hematopoiesis.

A large fraction of the hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) are deficient in membrane expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs). Current evidence suggests that this deficiency is sufficient to account for the hemolytic and thrombotic manifestations of this disease but not for its frequent association with aplastic anemia, an autoimmune disorder in which the patients' own hematopoietic progenitor cells are the target. Mutations in X-linked gene PIG-A, encoding one of several enzymes required for the biosynthesis of the glycophosphatidylinositol anchor, have been found in all PNH patients studied to date. Recent experiments with murine Pig-a knock-out embryonic stem cells show that although embryogenesis is critically dependent on normal GPI-AP expression, Pig-a-deficient cells can undergo apparently normal hematopoietic differentiation if they develop in a GPI-AP-replete environment. Thus, in an in vitro mouse model of PNH, Pig-a mutations confer no gross proliferative or differentiative advantage or disadvantage, suggesting an unidentified process selecting for these mutations in the bone marrow of patients with the PNH-aplastic anemia syndrome. The rescue of hematopoiesis observed in chimeric cultures of knock-out and normal cells was accompanied by intercellular transfer of GPI-AP, suggesting exciting new possibilities for future therapeutic manipulations in PNH patients.

Animals↗
PubMed1997

MYCN protein expression as a predictor of neuroblastoma prognosis.

About half of nonlocalized neuroblastomas have MYCN gene amplification and usually progress rapidly, but the half without such amplification also do poorly, albeit progressing more slowly. We hypothesize that overexpression of MYCN protein can occur without gene amplification and that this expression reliably predicts the prognosis of neuroblastoma. To determine whether MYCN expression correlated with outcome, we assayed MYCN protein immunohistochemically in 180 archival pretreatment and posttreatment samples and stratified the 57 conventionally treated stage IVS, III, and IV patients by these conventional prognostic factors: stage, age, serum ferritin, Shimada histology, urinary catecholamine ratio, and MYCN gene status. At a median follow-up of >/=6.8 years, we found in patients with known MYCN gene status that the 23 of 37 without gene amplification fared no better than the 14 of 37 with gene amplification (P = 0.35 and 0.21, comparing relapse-free and survival rates). Conversely, in patients without MYCN gene amplification, 9 of 23 were found to overexpress MYCN protein pretreatment, and they did worse than the 14 of 23 without detectable MYCN protein (P = 0.0016 and 0.022, comparing relapse-free and survival rates). Furthermore, MYCN protein expression was prognostic without (P = 0.00001) and with (P = 0.0007) stratifying all 57 patients by MYCN gene status, each conventional prognostic factor (P ranging from 0.00001-0.013), or simultaneously by the two most important factors, stage and age (P = 0.00076). We conclude that overexpression of MYCN protein without gene amplification correlated significantly with the clinical behavior of neuroblastoma and predicted outcome independently of other prognostic factors. This strongly supports the hypothesis that expression of the MYCN oncogene is critical for progression of neuroblastoma.

Adolescent↗
PubMed1996

Secondary and tertiary structure changes of reconstituted P-glycoprotein. A Fourier transform attenuated total reflection infrared spectroscopy analysis.

The structure of purified P-glycoprotein functionally reconstituted into liposomes was investigated by attenuated total reflection Fourier transform infrared spectroscopy. A quantitative evaluation of the secondary structure and a kinetic of 2H/H exchange of the P-glycoprotein were performed both in the presence and in the absence of MgATP, MgATP-verapamil, and MgADP. This approach was previously shown to be a useful tool to detect tertiary structure changes resulting from the interaction between a protein and its specific ligands, as established for the Neurospora crassa H+-ATPase. 2H/H exchange measurements provided evidence that a large fraction of the P-glycoprotein is poorly accessible to the aqueous medium. Addition of MgATP induced an increased accessibility to the solvent of a population of amino acids, while addition of MgATP-verapamil resulted in a subtraction of a part of the protein from access to the aqueous solvent. No significant changes were observed upon addition of MgADP or verapamil alone. The secondary structure of P-glycoprotein was not affected by addition of ligands. The variations observed in the 2H/H exchange rate when P-glycoprotein interacted with the above ligands therefore represented tertiary structure changes. Fluorescence quenching experiments confirmed that MgATP-induced changes are to be found in the tertiary structure of the enzyme.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Characterization and epitope mapping of several new anti-P-glycoprotein monoclonal antibodies.

Monoclonal antibodies (MAbs) were raised against partially purified Class I P-glycoprotein from multidrug-resistant Chinese hamster ovary CHRB30 cells. Fifteen stable monoclonal hybridoma cell lines were established, and the secreted antibodies were classified into 8 groups on the basis of banding pattern on immunoblots of P-glycoprotein digested with cyanogen bromide or partially digested with proteases. One representative of each group was tested further for several activities. Six of the 8 recognized human P-glycoprotein in the multidrug-resistant SKVLBI cell line. None of the antibodies recognized P-glycoprotein in unfixed cells, suggesting that all recognize cytoplasmic epitopes or extracellular epitopes not accessible in native P-glycoprotein. All 8 antibodies were able to immunoprecipitate P-glycoprotein from non-denaturing detergent solution. The linear epitopes of the antibodies were mapped to 11-27 amino acids. Two of the antibodies had epitopes in the linker region, 3 in the N-terminal nucleotide binding domain, 2 in the C-terminal nucleotide binding domain and 1 in the predicted cytoplasmic loop between predicted transmembrane helices 8 and 9. These antibodies, with known epitopes, could have uses for P-glycoprotein detection, structure/function studies, purification and quantitation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Duplication and evolution of the P-glycoprotein genes in pig.

The P-glycoproteins (Pgp's) are a small family of proteins frequently associated with the multidrug resistance phenotype in drug-selected cell lines. The number of Pgp isoforms in different mammalian species is variable although the reason for having a larger or smaller number of isoforms is not known. Two isoform classes from human, and three from rodents have been extensively characterised and have been shown to have independent expression patterns and substrate preferences. We have cloned 3' terminal genomic fragments for five members of the Pgp multigene family from the pig, which is the largest number of Pgp genes found in any mammalian species to date. Sequential duplications of one class of Pgp gene have given rise to this large gene family since four genes show similarity to the drug resistance-causing Class I isoform of Pgp. The fifth pig Pgp gene shows similarity to the phosphatidylcholine-translocating Class III isoform. The history of the duplications creating this large gene family can be traced by atypical features which have been inherited in common. These include a mutation in the stop codon at the 3' end of four Class I Pgp genes, increasing the coding region by six amino acids, and a SINE element of the PRE1 family inserted into the 3' untranslated region of three Class I Pgp's. We demonstrate expression of Class I pgp in pig brain cultured capillary endothelial cells, and Class III pgp in the liver, two important sites of expression of Pgp in rodents and humans. Thus there appears to be strong phylogenetic conservation in mammals of both sequence and expression of these two Pgp isoforms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Chemotherapy with focal therapy can cure intraocular retinoblastoma without radiotherapy.

BACKGROUND: External beam radiotherapy is standard treatment for medium and large, or visually threatening, intraocular retinoblastoma but markedly increases the risk of cosmetic deformities and second malignant neoplasms in children with germline RB1 mutations. Large tumors and those with vitreous seeds do poorly despite radiotherapy. Chemotherapy traditionally is ineffective for intraocular retinoblastoma, perhaps because many retinoblastomas overexpress the multidrug resistance protein, P-glycoprotein. OBJECTIVE: To avoid radiotherapy in the management of intraocular retinoblastoma by using chemotherapy and focal therapy. INTERVENTIONS: We shrank retinoblastomas in 40 eyes of 31 patients that conventionally should be enucleated or receive radiotherapy by using chemotherapy (ie, vincristine-teniposide, 8 eyes; additional carboplatin, 32 eyes) combined with the administration of cyclosporine, a known multidrug-resistance-reversal agent. We then consolidated these responses to chemotherapy by subsequent 532- and 1064-nm laser therapy and cryotherapy. RESULTS: At the median follow-up of 2 2/3 [corrected] years (range, 1/10-4 3/4 years), the results of treatment were excellent. The actuarial relapse-free rate was 89% in patients not previously treated (91% for 28 eyes) and 67% in patients treated after relapse from previous therapy (70% for 12 eyes). For the eyes with the worst prognosis (ie, vitreous seeds), the relapse-free rate was 88%, better than previously reported. Cyclosporine is nontoxic and did not enhance the expected toxic effects of chemotherapy. Most eyes required laser therapy, cryotherapy, or both for consolidation of tumor control. CONCLUSIONS: This pilot study suggests that most retinoblastomas are curable by combining chemotherapy with cyclosporine therapy, laser therapy, and cryotherapy, without requiring external beam radiotherapy. We propose a randomized trial to clarify the role of cyclosporine.

Antineoplastic Combined Chemotherapy Protocols↗