A novel LC-MS/MS-based assay for the simultaneous quantification of aldosterone-related steroids in human urine.

OBJECTIVES: Primary aldosteronism is the most common cause of endocrine hypertension and is associated with significant cardiovascular morbidities. The diagnostic workup depends on determinations of plasma aldosterone and renin which are highly variable and associated with false-positive and false-negative results. Quantification of aldosterone in 24 h urine may provide more reliable results, but the methodology is not well established. We aimed to establish an assay for urinary aldosterone and related steroids with suitability for clinical routine implementation. METHODS: Here, we report on the development and validation of a quantitative LC-MS/MS method for six urinary steroids: aldosterone, cortisol, 18-hydroxycorticosterone, 18-hydroxycortisol, 18-oxocortisol, tetrahydroaldosterone. After enzymatic deconjugation, total steroids were extracted using SepPak tC18 plates and quantified in positive electrospray ionization mode on a QTRAP 6500+ mass spectrometer. RESULTS: Excellent linearity was demonstrated with R2>0.998 for all analytes. Extraction recoveries were 89.8-98.4 % and intra- and inter-day coefficients of variations were <6.4 and <9.0 %, establishing superb precision. Patients with primary aldosteronism (n=10) had higher mean 24 h excretions of aldosterone-related metabolites than normotensive volunteers (n=20): 3.91 (95 % CI 2.27-5.55) vs. 1.92 (1.16-2.68) µmol/mol for aldosterone/creatinine, 2.57 (1.49-3.66) vs. 0.79 (0.48-1.10) µmol/mol for 18-hydroxycorticosterone/creatinine, 37.4 (13.59-61.2) vs. 11.61 (10.24-12.98) µmol/mol for 18-hydroxycortisol/creatinine, 1.56 (0.34-2.78) vs. 0.13 (0.09-0.17) µmol/mol for 18-oxocortisol/creatinine, and 21.5 (13.4-29.6) vs. 7.21 (4.88-9.54) µmol/mol for tetrahydroaldosterone/creatinine. CONCLUSIONS: The reported assay is robust and suitable for routine clinical use. First results in patient samples, though promising, require clinical validation in a larger sample set.

Confirmatory testing of primary aldosteronism with saline infusion test and LC-MS/MS.

OBJECTIVE: Saline infusion testing (SIT) for confirmation of primary aldosteronism (PA) is based on impaired aldosterone suppression in PA compared to essential hypertension (EH). In the past, aldosterone was quantified using immunoassays (IA). Liquid chromatography tandem mass spectrometry (LC-MS/MS) is increasingly used in clinical routine. We aimed at a method-specific aldosterone threshold for the diagnosis of PA during SIT and explored the diagnostic utility of steroid panel analysis. DESIGN: Retrospective cohort study of 187 paired SIT samples (2009-2018). Diagnosis of PA (n = 103) and EH (n = 84) was established based on clinical routine workup without using LC-MS/MS values. SETTING: Tertiary care center. METHODS: LC-MS/MS using a commercial steroid panel. Receiver operator characteristics analysis was used to determine method-specific cut-offs using a positive predictive value (PPV) of 90% as criterion. RESULTS: Aldosterone measured by IA was on average 31 ng/L higher than with LC-MS/MS. The cut-offs for PA confirmation were 54 ng/L for IA (sensitivity: 95%, 95% CI: 89.0-98.4; specificity: 87%, 95% CI: 77.8-93.3; area under the curve (AUC): 0.955, 95% CI: 0.924-0.986; PPV: 90%, 95% CI: 83.7-93.9) and 69 ng/L for LC-MS/MS (79%, 95% CI: 69.5-86.1; 89%, 95% CI: 80.6-95.0; 0.902, 95% CI: 0.857-0.947; 90%, 95% CI: 82.8-94.4). Other steroids did not improve SIT. CONCLUSIONS: Aldosterone quantification with LC-MS/MS and IA yields comparable SIT-cut-offs. Lower AUC for LC-MS/MS is likely due to the spectrum of disease in PA and previous decision making based on IA results. Until data of a prospective trial with clinical endpoints are available, the suggested cut-off can be used in clinical routine.

Steroidogenesis in the NCI-H295 Cell Line Model is Strongly Affected By Culture Conditions and Substrain.

CONTEXT: NCI-H295 cells are the most widely used model for adrenal steroidogenesis and adrenocortical carcinoma and have been used for decades in laboratories worldwide. However, reported steroidogenic properties differ considerably. OBJECTIVE: To evaluate heterogeneity of steroidogenesis among NCI-H295 cell strains, clarify the influence of culture media and test response to inhibitors of steroidogenesis by using liquid chromatography tandem mass spectrometry (LC-MS/MS). METHODS: NCI-H295 cells were obtained from two cell banks and cultivated in different media. An LC-MS/MS-based panel analysis of thirteen steroids was adapted for cell culture supernatant. Cells were treated with metyrapone, abiraterone and mitotane. RESULTS: Mineralocorticoid synthesis was strongly affected by passaging as reflected by reduction of aldosterone secretion from 0.158±0.006 to 0.017±0.001 µg/106 cells (p<0.05). Relevant differences were also found for cells from two vendors in terms of aldosterone secretion (0.180±0.001 vs. 0.09±0.002 µg/106 cells, p<0.05). Selection of medium strongly impacted on cortisol secretion with>4-fold difference (40.6±5.5 vs. 182.1±23 µg/106 cells) and reflected differential activation of the glucocorticoid pathway. Exposure to abiraterone, metyrapone and mitotane resulted in characteristic steroidogenic profiles consistent with known mechanism of drug action with considerable differences in metabolites upstream of the blocked enzyme. CONCLUSION: We demonstrate that steroid hormone secretion in NCI-H295 cells is strongly affected by the individual strain, passage and growing conditions. These factors should be taken into account in the evaluation of experiments analyzing steroid parameters directly or as surrogate parameters of cell viability.

Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction.

Conditions of impaired adrenal function and tissue destruction, such as in Addison's disease, and treatment resistance of adrenocortical carcinoma (ACC) necessitate improved understanding of the pathophysiology of adrenal cell death. Due to relevant oxidative processes in the adrenal cortex, our study investigated the role of ferroptosis, an iron-dependent cell death mechanism and found high adrenocortical expression of glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4) genes, key factors in the initiation of ferroptosis. By applying MALDI mass spectrometry imaging to normal and neoplastic adrenocortical tissue, we detected high abundance of arachidonic and adrenic acid, two long chain polyunsaturated fatty acids which undergo peroxidation during ferroptosis. In three available adrenal cortex cell models (H295R, CU-ACC1 and CU-ACC-2) a high susceptibility to GPX4 inhibition with RSL3 was documented with EC50 values of 5.7 × 10-8, 8.1 × 10-7 and 2.1 × 10-8 M, respectively, while all non-steroidogenic cells were significantly less sensitive. Complete block of GPX4 activity by RSL3 led to ferroptosis which was completely reversed in adrenal cortex cells by inhibition of steroidogenesis with ketoconazole but not by blocking the final step of cortisol synthesis with metyrapone. Mitotane, the only approved drug for ACC did not induce ferroptosis, despite strong induction of lipid peroxidation in ACC cells. Together, this report is the first to demonstrate extraordinary sensitivity of adrenal cortex cells to ferroptosis dependent on their active steroid synthetic pathways. Mitotane does not induce this form of cell death in ACC cells.

A Micellar Mitotane Formulation with High Drug-Loading and Solubility: Physico-Chemical Characterization and Cytotoxicity Studies in 2D and 3D In Vitro Tumor Models.

Adrenocortical carcinoma (ACC) is a rare tumor and prognosis is overall poor but heterogeneous. Mitotane (MT) has been used for treatment of ACC for decades, either alone or in combination with cytotoxic chemotherapy. Even at doses up to 6 g per day, more than half of the patients do not achieve targeted plasma concentration (14-20 mg L-1 ) even after many months of treatment due to low water solubility, bioavailability, and unfavorable pharmacokinetic profile. Here a novel MT nanoformulation with very high MT concentrations in physiological aqueous media is reported. The MT-loaded nanoformulations are characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, and powder X-ray diffraction which confirms the amorphous nature of the drug. The polymer itself does not show any cytotoxicity in adrenal and liver cell lines. By using the ACC model cell line NCI-H295 both in monolayers and tumor cell spheroids, micellar MT is demonstrated to exhibit comparable efficacy to its ethanol solution. It is postulated that this formulation will be suitable for i.v. application and rapid attainment of therapeutic plasma concentrations. In conclusion, the micellar formulation is considered a promising tool to alleviate major drawbacks of current MT treatment while retaining bioactivity toward ACC in vitro.

Hsp90 inhibition in adrenocortical carcinoma: Limited drug synergism with mitotane.

90 kDa heat shock proteins (Hsp90) act as protein chaperones and play a role in modulating endoplasmic reticulum (ER) stress. Hsp90 inhibitors are under clinical investigation as cancer treatment. Mitotane therapy of adrenocortical carcinoma (ACC) has been shown to act through lipid-induced ER-stress. To explore the potential of Hsp90 inhibitors in ACC as a single agent and in combination with mitotane, we analyzed two independent gene expression data sets of adrenal tumors in silico and treated the ACC cell line model NCI-H295 with Hsp90 inhibitors BIIB021 (B) and CCT18159 (C) alone and in combination with mitotane. ER-stress markers were monitored by immunoblotting. Drug synergism was quantified using the median effect model with cell viability as read-out. Cytosolic Hsp90 isoforms AA1 and AB1 were significantly overexpressed in ACC. Viability of H295 cells was impaired by B and C as single agents with an EC50 of 5.7 × 10-6M and 12.1 × 10-6M. B but not C dose-dependently increased XBP1 splicing and CHOP expression indicative of ER-stress activation. ER-stress marker expression was enhanced by co-incubation of B with 10  µM but not 5  µM mitotane. Maximal CHOP expression was induced by 25 µM mitotane alone with no additional effect of B. Combination indices (CI) of B and C with mitotane ranged from 0.64 to 1.38 and 0.68 to 1.30, respectively where CI values < 0.5 support clinically-relevant drug synergism. In conclusion, Hsp90 paralogs are differentially expressed in ACC and B but not C activates ER-stress in ACC cells. No meaningful drug synergism of Hsp90 inhibitors with mitotane was observed.

Plasma steroid metabolome profiling for the diagnosis of adrenocortical carcinoma.

Objective Current workup for the pre-operative distinction between frequent adrenocortical adenomas (ACAs) and rare but aggressive adrenocortical carcinomas (ACCs) combines imaging and biochemical testing. We here investigated the potential of plasma steroid hormone profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS) for the diagnosis of malignancy in adrenocortical tumors. Design Retrospective cohort study of prospectively collected EDTA-plasma samples in a single tertiary reference center. Methods Steroid hormone profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS) in random plasma samples and logistic regression modeling. Results Fifteen steroid hormones were quantified in 66 ACAs (29 males; M) and 42 ACC (15 M) plasma samples. Significantly higher abundances in ACC vs ACA were observed for 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, DHEA, DHEAS and estradiol (all P < 0.05). Maximal areas under the curve (AUC) for discrimination between ACA and ACC for single analytes were only 0.76 (estradiol) and 0.77 (progesterone), respectively. Logistic regression modeling enabled the discovery of diagnostic signatures composed of six specific steroids for male and female patients with AUC of 0.95 and 0.94, respectively. Positive predictive values in males and females were 92 and 96%, negative predictive values 90 and 86%, respectively. Conclusion This study in a large adrenal tumor patient cohort demonstrates the value of plasma steroid hormone profiling for diagnosis of ACC. Application of LC-MS/MS analysis and of our model may facilitate diagnosis of malignancy in non-expert centers. We propose to continuously evaluate and improve diagnostic accuracy of LC-MS/MS profiling by applying machine-learning algorithms to prospectively obtained steroid hormone profiles.

Abiraterone Acetate for Cushing Syndrome: Study in a Canine Primary Adrenocortical Cell Culture Model.

Abiraterone acetate (AA) is a potent inhibitor of steroidogenic enzyme 17α-hydroxylase/17,20-lyase (CYP17A1). AA is approved for the treatment of prostate cancer but could also be used to treat patients with Cushing syndrome (CS). Similar to humans, canine glucocorticoid synthesis requires CYP17A1, providing a useful animal model. The objective of this study was to preclinically investigate the effect of AA on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures (n = 9) from the adrenal glands of nine healthy dogs. The cells were incubated with AA (0.125 nM to 10 µM) for 72 hours under basal conditions and with 100 nM ACTH(1-24). Adrenocortical hormone concentrations were measured in culture medium using liquid chromatography-mass spectrometry, RNA was isolated from cells for subsequent real-time quantitative PCR analysis, and cell viability was assessed with an alamarBlue™ assay. AA reduced cortisol (IC50, 21.4 ± 4.6 nM) without affecting aldosterone under basal and ACTH-stimulated conditions. AA increased progesterone under basal and ACTH-stimulated conditions but reduced corticosterone under basal conditions, suggesting concurrent inhibition of 21-hydroxylation. AA did not affect the mRNA expression of steroidogenic enzymes and did not inhibit cell viability. In summary, primary canine adrenocortical cell culture is a useful model system for drug testing. For the treatment of CS, AA may to be superior to other steroidogenesis inhibitors due to its low toxicity. For future in vivo studies, dogs with endogenous CS may provide a useful animal model.

The Plant-Derived Naphthoquinone Droserone Inhibits In Vitro Measles Virus Infection.

The naphthoquinone droserone (1) is a natural product occurring in dicotyledonous plants. We have now observed that the addition of 1 during infection of tissue culture cells with measles virus considerably reduced the infection. Interestingly, the infection was inhibited only when droserone (1) was added during virus entry, but not when added to the cells prior to virus uptake or after virus uptake. These findings suggest that 1 interacts with viral particles to reduce infectivity. The formation of progeny measles virus particles was inhibited to 50 % by droserone (1) at a concentration (IC50) of approximately 2 µM with a half-maximal cytotoxicity (CC50) of about 60 µM for Vero cells. Other tested naphthoquinone derivatives, among them the likewise natural plumbagin (2), but also synthetic analogs, were either more cytotoxic or not as effective as 1. Thus, our data do not support the development of naphthoquinone derivatives into antiviral compounds, but suggest that they may be interesting research tools to study measles virus entry into cells.

Drug Synergism of Proteasome Inhibitors and Mitotane by Complementary Activation of ER Stress in Adrenocortical Carcinoma Cells.

Mitotane is the only drug approved for treatment of the orphan disease adrenocortical carcinoma (ACC) and was recently shown to be the first clinically used drug acting through endoplasmic reticulum (ER)-stress induced by toxic lipids. Since mitotane has limited clinical activity as monotherapy, we here study the potential of activating ER-stress through alternative pathways. The single reliable NCI-H295 cell culture model for ACC was used to study the impact MG132, bortezomib (BTZ) and carfilzomib (CFZ) on mRNA and protein expression of ER-stress markers, cell viability and steroid hormone secretion. We found all proteasome inhibitors alone to trigger expression of mRNA (spliced X-box protein 1, XBP1) and protein markers indicative of the inositol-requiring enzyme 1 (IRE1) dependent pathway of ER-stress but not phosphorylation of eukaryotic initiation factor 2α (eIF2α), a marker of the PRKR-like endoplasmic reticulum kinase (PERK)-dependent pathway. Whereas mitotane alone activated both pathways, combination of BTZ and CFZ with low-dose mitotane blocked mitotane-induced eIF2α phosphorylation but increased XBP1-mRNA splicing indicating that proteasome inhibitors can commit signalling towards a single ER-stress pathway in ACC cells. By applying the median effect model of drug combinations using cell viability as a read out, we determined significant drug synergism between mitotane and both BTZ and CFZ. In conclusion, combination of mitotane with activators of ER-stress through the unfolded protein response is synergistic in an ACC cell culture model. Since proteasome inhibitors are readily available clinically, they are attractive candidates to study for ACC treatment in clinical trials in combination with mitotane.

Association of mitotane with chylomicrons and serum lipoproteins: practical implications for treatment of adrenocortical carcinoma.

OBJECTIVE: Oral mitotane (o,p'-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC). AIM: Serum mitotane concentrations >14  mg/l are targeted for improved efficacy but not achieved in about half of patients. Here we aimed at a better understanding of intestinal absorption and lipoprotein association of mitotane and metabolites o,p'-dichlorodiphenylacetic acid (o,p'-DDA) and o,p'-dichlorodiphenyldichloroethane (o,p'-DDE). DESIGN: Lipoproteins were isolated by ultracentrifugation from the chyle of a 29-year-old patient and serum from additional 14 ACC patients treated with mitotane. HPLC was applied for quantification of mitotane and metabolites. We assessed NCI-H295 cell viability, cortisol production, and expression of endoplasmic reticulum (ER) stress marker genes to study the functional consequences of mitotane binding to lipoproteins. RESULTS: Chyle of the index patient contained 197  mg/ml mitotane, 53  mg/ml o,p'-DDA, and 51  mg/l o,p'-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3), and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction. DISCUSSION: Mitotane content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p'-DDE was similarly distributed, but 87.9±4.2% of o,p'-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti-proliferative and anti-hormonal effects on NCI-H295 cells and reduced ER stress marker gene expression. CONCLUSION: Mitotane absorption involves chylomicron binding. High concentrations of o,p'-DDA and o,p'-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane suggesting that lipoprotein-free mitotane is the therapeutically active fraction.

Mitotane Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid-Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells.

Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer.

Experimental adaptation of wild-type canine distemper virus (CDV) to the human entry receptor CD150.

Canine distemper virus (CDV), a close relative of measles virus (MV), is widespread and well known for its broad host range. When the goal of measles eradication may be achieved, and when measles vaccination will be stopped, CDV might eventually cross the species barrier to humans and emerge as a new human pathogen. In order to get an impression how fast such alterations may occur, we characterized required adaptive mutations to the human entry receptors CD150 (SLAM) and nectin-4 as first step to infect human target cells. Recombinant wild-type CDV-A75/17(red) adapted quickly to growth in human H358 epithelial cells expressing human nectin-4. Sequencing of the viral attachment proteins (hemagglutinin, H, and fusion protein, F) genes revealed that no adaptive alteration was required to utilize human nectin-4. In contrast, the virus replicated only to low titres (10(2) pfu/ml) in Vero cells expressing human CD150 (Vero-hSLAM). After three passages using these cells virus was adapted to human CD150 and replicated to high titres (10(5) pfu/ml). Sequence analyses revealed that only one amino acid exchange in the H-protein at position 540 Asp→Gly (D540G) was required for functional adaptation to human CD150. Structural modelling suggests that the adaptive mutation D540G in H reflects the sequence alteration from canine to human CD150 at position 70 and 71 from Pro to Leu (P70L) and Gly to Glu (G71E), and compensates for the gain of a negative charge in the human CD150 molecule. Using this model system our data indicate that only a minimal alteration, in this case one adaptive mutation, is required for adaptation of CDV to the human entry receptors, and help to understand the molecular basis why this adaptive mutation occurs.

The innate antiviral factor APOBEC3G targets replication of measles, mumps and respiratory syncytial viruses.

The cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) exerts antiviral activity against retroviruses, hepatitis B virus, adeno-associated virus and transposable elements. We assessed whether the negative-strand RNA viruses measles, mumps and respiratory syncytial might be affected by A3G, and found that their infectivity was reduced by 1-2 logs (90-99 %) in A3G overexpressing Vero cells, and in T-cell lines expressing A3G at physiological levels. Viral RNA was co-precipitated with HA-tagged A3G and could be amplified by RT-PCR. Interestingly, A3G reduced viral transcription and protein expression in infected cells by 50-70 %, and caused an increased mutation frequency of 0.95 mutations per 1000 nt in comparison to the background level of 0.22/1000. The observed mutations were not specific for A3G [cytidine to uridine (C→U) or guanine to adenine (G→A) hypermutations], nor specific for ADAR (adenosine deaminase acting on RNA, A→G and U→C transitions, with preference for next neighbour-nucleotides U = A>C>G). In addition, A3G mutants with inactivated catalytic deaminase (H257R and E259Q) were inhibitory, indicating that the deaminase activity is not required for the observed antiviral activity. In combination, impaired transcription and increased mutation frequencies are sufficient to cause the observed reduction in viral infectivity and eliminate virus replication within a few passages in A3G-expressing cells.

Clearance of measles virus from persistently infected cells by short hairpin RNA.

Subacute sclerosing panencephalitis (SSPE) is a demyelinating central nervous system disease caused by a persistent measles virus (MV) infection of neurons and glial cells. There is still no specific therapy available, and in spite of an intact innate and adaptive immune response, SSPE leads inevitably to death. In order to select effective antiviral short interfering RNAs (siRNAs), we established a plasmid-based test system expressing the mRNA of DsRed2 fused with mRNA sequences of single viral genes, to which certain siRNAs were directed. siRNA sequences were expressed as short hairpin RNA (shRNA) from a lentiviral vector additionally expressing enhanced green fluorescent protein (EGFP) as an indicator. Evaluation by flow cytometry of the dual-color system (DsRed and EGFP) allowed us to find optimal shRNA sequences. Using the most active shRNA constructs, we transduced persistently infected human NT2 cells expressing virus-encoded HcRed (piNT2-HcRed) as an indicator of infection. shRNA against N, P, and L mRNAs of MV led to a reduction of the infection below detectable levels in a high percentage of transduced piNT2-HcRed cells within 1 week. The fraction of virus-negative cells in these cultures was constant over at least 3 weeks posttransduction in the presence of a fusion-inhibiting peptide (Z-Phe-Phe-Gly), preventing the cell fusion of potentially cured cells with persistently infected cells. Transduced piNT2 cells that lost HcRed did not fuse with underlying Vero/hSLAM cells, indicating that these cells do not express viral proteins any more and are "cured." This demonstrates in tissue culture that NT2 cells persistently infected with MV can be cured by the transduction of lentiviral vectors mediating the long-lasting expression of anti-MV shRNA.