RESUMEN
Liquid biopsy in cancer has gained momentum in clinical research and is experiencing a boom for a variety of applications. There are significant efforts to utilize liquid biopsies in cancer for early detection and treatment stratification, as well as residual disease and recurrence monitoring. Although most efforts have used circulating tumor cells and circulating tumor DNA for this purpose, exosomes and other extracellular vesicles have emerged as a platform with potentially broader and complementary applications. Exosomes/extracellular vesicles are small vesicles released by cells, including cancer cells, into the surrounding biofluids. These exosomes contain tumor-derived materials such as DNA, RNA, protein, lipid, sugar structures, and metabolites. In addition, exosomes carry molecules on their surface that provides clues regarding their origin, making it possible to sort vesicle types and enrich signatures from tissue-specific origins. Exosomes are part of the intercellular communication system and cancer cells frequently use them as biological messengers to benefit their growth. Since exosomes are part of the disease process, they have become of tremendous interest in biomarker research. Exosomes are remarkably stable in biofluids, such as plasma and urine, and can be isolated for clinical evaluation even in the early stages of the disease. Exosome-based biomarkers have quickly become adopted in the clinical arena and the first exosome RNA-based prostate cancer test has already helped >50 000 patients in their decision process and is now included in the National Comprehensive Cancer Network guidelines for early prostate cancer detection. This review will discuss the advantages and challenges of exosome-based liquid biopsies for tumor biomarkers and clinical implementation in the context of circulating tumor DNA and circulating tumor cells.
Asunto(s)
ADN Tumoral Circulante , Exosomas , Células Neoplásicas Circulantes , Biomarcadores de Tumor , Humanos , Biopsia Líquida , Masculino , Recurrencia Local de NeoplasiaRESUMEN
Previous analysis has refined the location of the gene for Norrie disease, a severe, X-linked, recessive neurodevelopmental disorder, to a yeast artificial chromosome subfragment of 160 kilobases (kb). This fragment was used to screen cDNA libraries from human fetal and adult retina. As a result, we have identified an evolutionarily conserved cDNA, which is expressed in fetal and adult brain and encodes a predicted protein of 133 amino acids. The cDNA detects genomic sequences which span a maximum of 50 kb, and which are partly deleted in several typical Norrie disease patients. An EcoRI polymorphism with a calculated heterozygosity value of 43% was observed. The locus identified is a strong candidate for the Norrie disease gene.
Asunto(s)
Ceguera/genética , Cromosoma X , Secuencia de Aminoácidos , Secuencia de Bases , Ceguera/congénito , Mapeo Cromosómico , Cromosomas Fúngicos , ADN/genética , Análisis Mutacional de ADN , Biblioteca de Genes , Ligamiento Genético , Humanos , Masculino , Datos de Secuencia Molecular , Eliminación de SecuenciaRESUMEN
Familial exudative vitreoretinopathy (FEVR) is a hereditary disorder characterized by an abnormality of the peripheral retina. Both autosomal dominant (adFEVR) and X-linked (XLFEVR) forms have been described, but the biochemical defect(s) underlying the symptoms are unknown. Molecular analysis of the Norrie gene locus (NDP) in a four generation FEVR family (shown previously to exhibit linkage to the X-chromosome markers DXS228 and MAOA (Xp11.4-p11.3)) reveals a missense mutation in the highly conserved region of the NDP gene, which caused a neutral amino acid substitution (Leu124Phe), was detected in all of the affected males, but not in the unaffected family members, nor in normal controls. The observations suggest that phenotypes of both XLFEVR and Norrie disease can result from mutations in the same gene.
Asunto(s)
Proteínas del Ojo/genética , Mutación , Proteínas del Tejido Nervioso/genética , Enfermedades de la Retina/genética , Cuerpo Vítreo , Cromosoma X , Adolescente , Secuencia de Bases , Niño , ADN Complementario , Oftalmopatías/genética , Femenino , Ligamiento Genético , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Retina/anomalías , Cuerpo Vítreo/anomalíasRESUMEN
Familial dysautonomia (DYS), the Riley-Day syndrome, is an autosomal recessive disorder characterized by developmental loss of neurons from the sensory and autonomic nervous system. It is limited to the Ashkenazi Jewish population, where the carrier frequency is 1 in 30. We have mapped the DYS gene to chromosome 9q31-q33 by linkage with ten DNA markers in 26 families. The maximum lod score of 21.1 with no recombinants was achieved with D9S58. This marker also showed strong linkage disequilibrium with DYS, with one allele present on 73% of affected chromosomes compared to 5.4% of controls (chi 2 = 3142, 15 d.f. p < 0.0001). D9S53 and D9S105 represent the closest flanking markers for the disease gene. This localization will permit prenatal diagnosis of DYS in affected families and aid the isolation of the disease gene.
Asunto(s)
Cromosomas Humanos Par 9 , Disautonomía Familiar/genética , Marcadores Genéticos , Polimorfismo Genético , Alelos , Mapeo Cromosómico , Disautonomía Familiar/diagnóstico , Disautonomía Familiar/epidemiología , Disautonomía Familiar/etnología , Femenino , Enfermedades Fetales/diagnóstico , Enfermedades Fetales/genética , Frecuencia de los Genes , Genes Recesivos , Tamización de Portadores Genéticos , Humanos , Incidencia , Judíos/genética , Desequilibrio de Ligamiento , Escala de Lod , Masculino , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Diagnóstico PrenatalRESUMEN
Early-onset torsion dystonia is a movement disorder, characterized by twisting muscle contractures, that begins in childhood. Symptoms are believed to result from altered neuronal communication in the basal ganglia. This study identifies the DYT1 gene on human chromosome 9q34 as being responsible for this dominant disease. Almost all cases of early-onset dystonia have a unique 3-bp deletion that appears to have arisen idependently in different ethnic populations. This deletion results in loss of one of a pair of glutamic-acid residues in a conserved region of a novel ATP-binding protein, termed torsinA. This protein has homologues in nematode, rat, mouse and humans, with some resemblance to the family of heat-shock proteins and Clp proteases.
Asunto(s)
Cromosomas Humanos Par 9 , Distonía Muscular Deformante/genética , Chaperonas Moleculares , Transportadoras de Casetes de Unión a ATP/genética , Edad de Inicio , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Mapeo Cromosómico , Análisis Mutacional de ADN , Tamización de Portadores Genéticos , Ligamiento Genético , Marcadores Genéticos , Humanos , Judíos/genética , Linfocitos , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Ratas , Proteínas Recombinantes/biosíntesis , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Adeno-associated virus (AAV)-mediated gene replacement for lysosomal disorders have been spurred by the ability of some serotypes to efficiently transduce neurons in the brain and by the ability of lysosomal enzymes to cross-correct among cells. Here, we explored enzyme replacement therapy in a knock-out mouse model of congenital neuronal ceroid lipofuscinosis (NCL), the most severe of the NCLs in humans. The missing protease in this disorder, cathepsin D (CathD) has high levels in the central nervous system. This enzyme has the potential advantage for assessing experimental therapy in that it can be imaged using a near-infrared fluorescence (NIRF) probe activated by CathD. Injections of an AAV2/rh8 vector-encoding mouse CathD (mCathD) into both cerebral ventricles and peritoneum of newborn knock-out mice resulted in a significant increase in lifespan. Successful delivery of active CathD by the AAV2/rh8-mCathD vector was verified by NIRF imaging of mouse embryonic fibroblasts from knock-out mice in culture, as well as by ex vivo NIRF imaging of the brain and liver after gene transfer. These studies support the potential effectiveness and imaging evaluation of enzyme replacement therapy to the brain and other organs in CathD null mice via AAV-mediated gene delivery in neonatal animals.
Asunto(s)
Catepsina D/genética , Colorantes Fluorescentes , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Rayos Infrarrojos , Lipofuscinosis Ceroideas Neuronales/terapia , Animales , Animales Recién Nacidos , Química Encefálica , Dependovirus/genética , Modelos Animales de Enfermedad , Terapia de Reemplazo Enzimático/métodos , Vectores Genéticos , Hígado/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Lipofuscinosis Ceroideas Neuronales/genéticaRESUMEN
Herein, we describe a novel approach in the search for prostate cancer biomarkers, which relies on the transcriptome within tumour exosomes. As a proof-of-concept, we show the presence of two known prostate cancer biomarkers, PCA-3 and TMPRSS2:ERG the in exosomes isolated from urine of patients, showing the potential for diagnosis and monitoring cancer patients status.
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Biomarcadores de Tumor/orina , Exosomas/patología , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/orina , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/orina , Biomarcadores de Tumor/genética , Exosomas/genética , Humanos , Masculino , Proteínas de Fusión Oncogénica/análisis , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/orina , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/análisis , Urinálisis/métodosRESUMEN
A defective herpes simplex virus 1 (HSV-1) vector, pHSVlac, has been developed that contains a transcription unit that places the Escherichia coli lacZ gene under the control of the HSV-1 immediate early 4/5 promoter. The vector pHSVlac was propagated with the HSV-1 temperature-sensitive mutant ts K as helper virus. Infection of neurons from rat superior cervical ganglia and dorsal root ganglia in primary culture resulted in stable expression of high levels of beta-galactosidase without cell death. These HSV-1 vectors should be useful for introducing genes into postmitotic cells, such as neurons, in vitro and in vivo.
Asunto(s)
Virus Defectuosos/genética , Escherichia coli/genética , Galactosidasas/genética , Vectores Genéticos , Neuronas/microbiología , beta-Galactosidasa/genética , Animales , Células Cultivadas , ADN Viral/metabolismo , Escherichia coli/enzimología , Técnica del Anticuerpo Fluorescente , Virus Helper , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Simplexvirus/genética , Transfección , beta-Galactosidasa/biosíntesisRESUMEN
The Lesch-Nyhan syndrome in humans is characterized by lack of hypoxanthine phosphoribosyltransferase activity and neurologic abnormalities that suggest changes in catecholamine metabolism. Monoamine oxidase, which degrades biogenic amines, has decreased activity in noradrenergic murine neuroblastoma cell lines lacking hypoxanthine phosphoribosyltransferase activity and in skin fibroblasts from patients with the Lesch-Nyhan syndrome.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/deficiencia , Síndrome de Lesch-Nyhan/enzimología , Monoaminooxidasa/metabolismo , Adolescente , Adulto , Catecol O-Metiltransferasa/metabolismo , Línea Celular , Niño , Dopamina beta-Hidroxilasa/metabolismo , Femenino , Fibroblastos , Humanos , Masculino , NeuronasRESUMEN
Fibroblasts were genetically modified to secrete nerve growth factor (NGF) by infection with a retroviral vector and then implanted into the brains of rats that had surgical lesions of the fimbria-fornix. The grafted cells survived and produced sufficient NGF to prevent the degeneration of cholinergic neurons that would die without treatment. In addition, the protected cholinergic cells sprouted axons that projected in the direction of the cellular source of NGF. These results indicate that a combination of gene transfer and intracerebral grafting may provide an effective treatment for some disorders of the central nervous system.
Asunto(s)
Encéfalo/patología , Fibroblastos/trasplante , Factores de Crecimiento Nervioso/fisiología , Acetilcolinesterasa/metabolismo , Animales , Encéfalo/citología , Encéfalo/enzimología , Supervivencia Celular , ADN/genética , Fibroblastos/metabolismo , Vectores Genéticos , Histocitoquímica , Virus de la Leucemia Murina de Moloney/genética , Factores de Crecimiento Nervioso/genética , RatasRESUMEN
Genetic and metabolic studies have been done on a large kindred in which several males are affected by a syndrome of borderline mental retardation and abnormal behavior. The types of behavior that occurred include impulsive aggression, arson, attempted rape, and exhibitionism. Analysis of 24-hour urine samples indicated markedly disturbed monoamine metabolism. This syndrome was associated with a complete and selective deficiency of enzymatic activity of monoamine oxidase A (MAOA). In each of five affected males, a point mutation was identified in the eighth exon of the MAOA structural gene, which changes a glutamine to a termination codon. Thus, isolated complete MAOA deficiency in this family is associated with a recognizable behavioral phenotype that includes disturbed regulation of impulsive aggression.
Asunto(s)
Agresión , Genes , Discapacidad Intelectual/genética , Monoaminooxidasa/genética , Mutación Puntual , Línea Celular , Células Cultivadas , Femenino , Humanos , Discapacidad Intelectual/enzimología , Masculino , Monoaminooxidasa/deficiencia , Linaje , Fenotipo , Piel/enzimología , Síndrome , Cromosoma XRESUMEN
TorsinA is the causative protein in the human neurologic disease early onset torsin dystonia, a movement disorder involving dysfunction in the basal ganglia without apparent neurodegeneration. Most cases result from a dominantly acting three-base pair deletion in the TOR1A gene causing loss of a glutamic acid near the carboxyl terminus of torsinA. Torsins are members of the AAA(+) superfamily of ATPases and are present in all multicellular organisms. Initial studies suggest that torsinA is an ER protein involved in chaperone functions and/or membrane movement.
Asunto(s)
Proteínas Portadoras/genética , Distonía Muscular Deformante/genética , Chaperonas Moleculares , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Distonía Muscular Deformante/fisiopatología , Humanos , Modelos Moleculares , Familia de Multigenes , Filogenia , Conformación Proteica , Eliminación de SecuenciaAsunto(s)
Dopamina/deficiencia , Distonía/metabolismo , GTP Ciclohidrolasa/deficiencia , Enfermedad de Parkinson/metabolismo , Edad de Inicio , Biopterinas/biosíntesis , Biopterinas/deficiencia , Mapeo Cromosómico , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 9 , Distonía/clasificación , Distonía/epidemiología , Distonía/genética , GTP Ciclohidrolasa/genética , Genes Dominantes , Genes Recesivos , Humanos , Japón/epidemiología , Judíos/genética , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/genética , Fenilalanina/sangre , Síndrome , Tirosina 3-Monooxigenasa/metabolismo , Cromosoma XRESUMEN
Schwannomas are benign tumors composed of dedifferentiated Schwann cells that form along peripheral nerves causing nerve compression often associated with pain and loss of function. Current surgical therapy involves total or subtotal surgical removal of the tumor, which may cause permanent nerve damage. In the present study, we explore an alternate means of therapy in which schwannomas are injected with a replication-conditional herpes simplex virus (HSV) vector to shrink the tumor through cell lysis during virus propagation. The oncolytic vector used, G47Delta, has deletions in HSV genes, which allow it to replicate selectively in dividing cells, sparing neurons. Two schwannoma cell lines were used to generate subcutaneous tumors in nude mice: HEI193, an immortalized human line previously established from an NF2 patient and NF2S-1, a newly generated spontaneous mouse line. Subcutaneous HEI193 tumors grew about ten times as fast as NF2S-1 tumors, and both regressed substantially following injection of G47Delta. Complete regression of HEI193 tumors was achieved in most animals, whereas all NF2S-1 tumors resumed growth within 2 weeks after vector injection. These studies provide a new schwannoma model for testing therapeutic strategies and demonstrate that oncolytic HSV vectors can be successfully used to shrink growing schwannomas.
Asunto(s)
Modelos Animales de Enfermedad , Ratones , Neurilemoma/terapia , Tumores Neuroendocrinos/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos , Simplexvirus , Animales , Línea Celular Tumoral , Eliminación de Gen , Humanos , Ratones Endogámicos , Trasplante de Neoplasias , Neurilemoma/patología , Tumores Neuroendocrinos/patología , Virus Oncolíticos/genética , Simplexvirus/genéticaRESUMEN
Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and radiation sensitivity. Herpes simplex virus type 1 (HSV-1) amplicon vectors provide a means to deliver large genes to the nervous system efficiently and safely. We have generated an amplicon vector, carrying human FLAG-tagged A-T mutated (ATM), as well as an enhanced green fluorescent protein (EGFP) marker gene. Due to the lack of effective and reliable antibodies for ATM and FLAG appropriate for immunohistochemistry in mouse tissue sections, expression of the human FLAG-tagged ATM was confirmed in the mouse cerebellum at the RNA level by reverse transcription followed by quantitative PCR, and by radioactive in situ hybridization. In addition, we were able to immunoprecipitate the full-length human ATM protein from the cerebella of Atm -/- mice post-infection. This vector has been injected into the cerebella of Atm -/- mice with gene delivery to thousands of cells, including Purkinje cells, based on the EGFP marker gene. The expression of human FLAG-tagged ATM has been demonstrated in the cerebella of Atm-/- mice at the transcription and translational level three days post-infection. To our knowledge, this is the first report of vector-mediated delivery of the human ATM cDNA to an Atm -/- mouse. These vectors provide the groundwork to develop gene therapy approaches for A-T patients.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cerebelo/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Expresión Génica/fisiología , Herpesvirus Humano 1/fisiología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting/métodos , Línea Celular , Cerebelo/virología , Técnicas de Transferencia de Gen , Vectores Genéticos/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación/métodos , Hibridación in Situ/métodos , Ratones , Ratones Noqueados , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Familial, early onset, generalized torsion dystonia is the most common and severe primary dystonia. The majority of cases are caused by a 3-bp deletion (GAG) in the coding region of the DYT1 (TOR1A) gene. The cellular and regional distribution of torsinA protein, which is restricted to neuronal cells and present in all brain regions by the age of 2 months has been described recently in human developing brain. TorsinB is a member of the same family of proteins and is highly homologous with its gene adjacent to that for torsinA on chromosome 9q34. TorsinA and torsinB share several remarkable features suggesting that they may interact in vivo. This study examined the expression of torsinB in the human brain of fetuses, infants and children up to 7 years of age. Our results indicate that torsinB protein expression is temporarily and spatially regulated in a similar fashion as torsinA. Expression of torsinB protein was detectable beginning at four to 8 weeks of age in the cerebellum (Purkinje cells), substantia nigra (dopaminergic neurons), hippocampus and basal ganglia and was predominantly restricted to neuronal cells. In contrast to torsinA, torsinB immunoreactivity was found more readily in the nuclear envelope. High levels of torsinB protein were maintained throughout infancy, childhood and adulthood suggesting that torsinB is also needed for developmental events occurring in the early postnatal phase and is necessary for functional activity throughout life.
Asunto(s)
Química Encefálica/fisiología , Encéfalo/crecimiento & desarrollo , Chaperonas Moleculares/biosíntesis , Neuronas/metabolismo , Adulto , Axones/metabolismo , Ganglios Basales/metabolismo , Western Blotting , Cerebelo/metabolismo , Niño , Preescolar , Citoplasma/metabolismo , Dendritas/metabolismo , Femenino , Hipocampo/metabolismo , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Masculino , Mesencéfalo/metabolismo , EmbarazoAsunto(s)
Trasplante de Tejido Encefálico/efectos adversos , Trasplante de Tejido Encefálico/métodos , Trasplante Heterólogo , Animales , Neoplasias Encefálicas/terapia , Trasplante de Tejido Encefálico/fisiología , Ensayos Clínicos como Asunto , Trasplante de Tejido Fetal/efectos adversos , Trasplante de Tejido Fetal/métodos , Trasplante de Tejido Fetal/fisiología , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos , Humanos , Ratones , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/terapia , Retroviridae/genética , Retroviridae/patogenicidad , Factores de Riesgo , Porcinos , Timidina Quinasa/genéticaRESUMEN
BACKGROUND: Although antifolates are popular agents for use in chemotherapy, they display minimal toxicity against slow-growing tumors and are toxic to actively replicating cells in normal tissues. These drugs are converted intracellularly into polyglutamate derivatives by the enzyme folylpolyglutamyl synthetase (FPGS). Because tumors with high expression of FPGS often respond to nontoxic antifolate doses, we investigated whether augmenting tumoral FPGS activity by gene delivery would enhance tumoral antifolate sensitivity. METHODS: 9L rat gliosarcoma cells were stably transfected with a human FPGS complementary DNA (cDNA), producing 9L/FPGS cells. The sensitivity of these cells to the antifolates methotrexate and edatrexate was measured in culture and in subcutaneous tumors, as was their ability to increase the chemosensitivity of nearby nontransfected cells, i.e., a bystander effect. The antifolate sensitivity of nonselected cells transduced with a hybrid amplicon vector that expressed FPGS was also ascertained. RESULTS: In comparison with 9L cells, 9L/FPGS cells displayed enhanced sensitivity to 4-hour pulses of antifolate. Subcutaneous 9L/FPGS tumors responded as well to methotrexate given every third day as 9L tumors did to daily treatment. A modest bystander effect was observed with edatrexate treatment in culture and in vivo. The observed bystander effect appeared to result from the release of antifolates by transfected cells after the removal of extracellular drug. In culture, enhanced antifolate sensitivity was also seen in other stably transfected rodent and human glioma cell lines, including one with high pre-existing FPGS activity, and in canine and human glioblastoma cell lines transduced with a vector bearing FPGS cDNA. CONCLUSIONS: FPGS gene delivery enhances the antifolate sensitivity of several glioma cell lines and merits further evaluation as a therapeutic strategy.
Asunto(s)
Aminopterina/análogos & derivados , Antimetabolitos Antineoplásicos/farmacología , Antagonistas del Ácido Fólico/farmacología , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Metotrexato/farmacología , Neoplasias/terapia , Péptido Sintasas/genética , Aminopterina/farmacología , Animales , Cromatografía en Capa Delgada , Perros , Relación Dosis-Respuesta a Droga , Glioma , Humanos , Neoplasias/enzimología , Neoplasias/genética , Péptido Sintasas/metabolismo , Ratas , Transducción Genética , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND: A bacterial enzyme, Escherichia coli cytosine deaminase, which converts the prodrug 5-fluorocytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. METHODS: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli cytosine deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou-Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. RESULTS AND CONCLUSIONS: The presence of transfected cytosine deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment.