ABSTRACT
Mxi1 is a Mad family member that plays a role in cell proliferation and differentiation. To test the role of Mxi1 on tumorigenesis of glioma cells we transfected a CMV-driven MXI1 cDNA in U87 human glioblastoma cells. Two clones were isolated expressing MXI1 levels 18- and 3.5-fold higher than wild-type U87 cells (clone U87.Mxi1.14 and U87.Mxi1.22, respectively). In vivo, U87.Mxi1.14 cells were not tumorigenic in nude mice and delayed development of tumours was observed with U87.Mxi1.22 cells. In vitro, the proliferation rate was partially and strongly inhibited in U87.Mxi1.22 and U87.Mxi1.14 cells respectively. The cell cycle analysis revealed a relevant accumulation of U87.Mxi1.14 cells in the G(2)/M phase. Interestingly, the expression of cyclin B1 was inhibited to about 60% in U87.Mxi1.14 cells. This inhibition occurs at the transcriptional level and depends, at least in part, on the E-box present on the cyclin B1 promoter. Consistent with this, the endogenous Mxi1 binds this E-box in vitro. Thus, our findings indicate that Mxi1 can act as a tumour suppressor in human glioblastomas through a molecular mechanism involving the transcriptional down-regulation of cyclin B1 gene expression.
Subject(s)
Cell Differentiation/physiology , Cell Division/drug effects , Cell Division/physiology , Cyclin B/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors , Cell Cycle/physiology , Cyclin B1 , DNA-Binding Proteins/genetics , G2 Phase , Genes, Reporter , Glioma , Humans , Kinetics , Mitosis , Recombinant Proteins/pharmacology , Time Factors , Transcription Factors/genetics , Transcription, Genetic , Transfection , Tumor Cells, Cultured , Tumor Suppressor ProteinsABSTRACT
Androgens control both growth and differentiation of the normal prostate gland. However, the mechanisms by which androgens act upon the cell cycle machinery to regulate these two fundamental processes are largely unknown. The cyclin-dependent kinase (cdk) inhibitor p27 is a negative cell cycle regulator involved in differentiation-associated growth arrest. Here, we investigate the role and regulation of p27 in the testosterone proprionate (TP)-stimulated regeneration of the ventral prostate (VP) of castrated rats. Continuous TP administration to castrated rats triggered epithelial cell proliferation, which peaked at 72 h, and then declined despite further treatment. Castration-induced atrophy of the VP was associated with a significant increase in p27 expression as compared with the VP of intact animals. Twelve hours after the initiation of androgen treatment, total p27 levels as well as its fraction bound to cdk2, its main target, significantly dropped in the VP of castrated rats. Thereafter, concomitantly to the induction of epithelial cell proliferation, the glandular morphology of VP was progressively restored at 48-96 h of TP treatment. During this period of the regenerative process, whereas both proliferating basal and secretory epithelial cells did not express p27, the protein was selectively up-regulated in the nonproliferating secretory epithelial compartment. This up-regulation of p27 expression was coincident with an increase in its association with, and presumably inhibition of, cdk2. At each time point of TP treatment, p27 abundance in the VP was inversely correlated with the level of its proteasome-dependent degradation activity measured in vitro in VP lysates, whereas only slight changes in the amount of p27 transcripts were detected. In addition, the antiandrogen flutamide blocked maximal TP-induced p27 degradation completely. Finally, the expression of skp2, the ubiquitin ligase that targets p27 for degradation, was seen to increase with androgen administration, preceding maximal proliferation and concomitantly to augmented p27 degradation activity. Taken together, our data indicate that androgens mediate both proliferation and differentiation signals in normal prostate epithelial cells in vivo, through regulation of p27.
Subject(s)
Cell Cycle Proteins , Microtubule-Associated Proteins/physiology , Prostate/cytology , Testosterone/pharmacology , Tumor Suppressor Proteins , Androgen Antagonists/pharmacology , Animals , Blotting, Western , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Division/drug effects , Cell Division/physiology , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/biosynthesis , Epithelial Cells/cytology , Epithelial Cells/drug effects , Fluorescent Antibody Technique , Flutamide/pharmacology , G1 Phase/drug effects , G1 Phase/physiology , Gene Expression Regulation/drug effects , In Situ Hybridization , Kinetics , Male , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Orchiectomy , Precipitin Tests , Prostate/drug effects , Prostate/physiology , Rats , Up-RegulationABSTRACT
Overexpression of interleukin 4 (IL-4) can impair the tumorigenicity of glioma cells, but direct evidence of its antitumor efficacy after in vivo gene transfer into malignant gliomas has not been provided. To test this, we first injected into the brain of Sprague Dawley rats a 1:1 mixture of C6 rat glioblastoma cells and psi2.L4SN20 or E86.L4SN50 retroviral producer cells (RPCs), secreting 20 and 50 ng of IL-4/5 x 10(5) cells/48 h, respectively. Twenty-seven and 56% of rats receiving injections with these low- or medium-level IL-4 RPCs, respectively, survived tumor injection, whereas control rats died in about 1 month. E86.L4SN50 RPCs coinjected with 9L gliosarcoma cells into syngeneic Fischer 344 rats yielded similar results. A novel IL-4 RPC clone expressing higher levels of IL-4, E86.L4SN200, coinjected with 9L cells increased to 75% the fraction of long-term survivors and induced tumor regression in 50% of rats when injected into established 9L gliosarcomas. Cured rats developed an immunological memory because they rejected a challenge of wild-type 9L cells into the contralateral hemisphere. Magnetic resonance imaging was used to monitor 9L and C6 gliomas and gave direct evidence for tumor rejection in treated rats. Immunohistology showed inflammatory infiltrates in IL-4-treated tumors in which CD8+ T lymphocytes were more abundant, although CD4+ T lymphocytes, B lymphocytes, and macrophages were also present. Overall, these findings suggest that IL-4 gene transfer is a new, promising approach for treating malignant gliomas.
Subject(s)
Brain Neoplasms/therapy , Genetic Therapy/methods , Glioma/therapy , Interleukin-4/genetics , Animals , Brain Neoplasms/immunology , Brain Neoplasms/metabolism , CD8-Positive T-Lymphocytes/immunology , DNA, Complementary/administration & dosage , DNA, Complementary/genetics , DNA, Complementary/metabolism , Female , Glioma/immunology , Glioma/metabolism , Interleukin-4/biosynthesis , Interleukin-4/immunology , Mice , Rats , Rats, Inbred F344 , Rats, Sprague-Dawley , Retroviridae/geneticsABSTRACT
In tumors, gap junctional intercellular communication (GJIC) is usually down-regulated and the expression of connexins, membrane proteins constituting gap junction channels, is often low or altered. GJIC, allowing the intercellular diffusion of ganciclovir (GCV) triphosphate, is also one mediator of the 'bystander effect', the phenomenon by which herpes simplex virus thymidine kinase (HSVtk)-transduced, neoplastic cells kill surrounding HSVtk-negative cells when treated with GCV. We set up experiments to evaluate the effects of retrovirus-mediated in vivo gene transfer of connexin 43 in malignancies with low GJIC capacity. We found that U-87 human glioblastoma cells transfected in vitro by the human Cx43 cDNA grow significantly more slowly than control U-87 cells and lose their tumorigenicity when injected subcutaneously in nude mice. When the Cx43 gene was transduced in vitro in U-87 cells by a retroviral producer cell line (N3.2.ii, titer 1.5 x 10(6) c.f.u./ml) in vivo results were similar. However, only when U-87 cells were co-injected with N3.2.ii cells in nude mice in a 1:5 ratio, a 50% reduction in tumor size was obtained during the first 3 weeks. Moreover the coinjection of U-87 cells with N3.2.ii and SBA cells (a retroviral producer cell line expressing the HSVtk gene), was not able to potentiate the effects of GCV administration, suggesting that Cx43 gene transfer requires more efficient vectors to increase the bystander effect in vivo.
Subject(s)
Connexin 43/genetics , Gene Transfer Techniques , Genetic Therapy/methods , Glioma/therapy , Retroviridae/genetics , Animals , Antiviral Agents/therapeutic use , Female , Ganciclovir/therapeutic use , Genetic Vectors , Glioma/drug therapy , Mice , Mice, Nude , Thymidine Kinase/genetics , Tumor Cells, CulturedSubject(s)
Brain Neoplasms/therapy , Gene Transfer Techniques , Glioma/therapy , Interleukin-4/genetics , Neurons/cytology , Neurons/transplantation , Animals , Brain Neoplasms/pathology , Cell Line , Corpus Striatum/cytology , DNA, Neoplasm/biosynthesis , DNA, Neoplasm/drug effects , Embryo, Mammalian , Female , Glioma/pathology , Interleukin-4/biosynthesis , Interleukin-4/pharmacology , Magnetic Resonance Imaging , Rats , Rats, Sprague-Dawley , Recombinant Proteins/biosynthesis , Recombinant Proteins/pharmacology , Retroviridae/genetics , Tumor Cells, CulturedABSTRACT
The growth of U-87 or C6 gliomas co-implanted in nude mice with retroviral producer cells (VPC) expressing the herpes simplex virus-thymidine kinase (HSV-tk) gene is only partially impaired by treatment with ganciclovir (GCV). The effect of GCV is even less evident when C6 and VPC are co-implanted into the rat brain. Furthermore, tumors from C6 cells carrying the HSV-tk gene are not eradicated by GCV, although they remain sensitive to GCV when replated in vitro. These limits of the HSV-tk/GCV system in glioma gene therapy may be due to insufficient gene transfer and/or insufficient delivery of GCV to glioma cells. Combination of HSV-tk and one or more cytokines may improve the antitumor efficacy. Among cytokines, interleukin-4 (IL-4) has already been shown to be active against gliomas. In nude mice, GCV treatment inhibited tumor growth more effectively after co-injection of C6 cells with a mixture of VPC transducing IL-4 and HSV-tk genes than after co-injection with either IL-4 or HSV-tk VPC only. In immunocompetent Sprague-Dawley rats, co-injection of IL-4 VPC and C6 cells was also effective in inhibiting the growth of C6 brain tumors, 38% of the animals surviving for at least 2 months. Furthermore, increased and prolonged antitumor efficacy was obtained by transducing both IL-4 and HSV-tk genes.
Subject(s)
Antimetabolites/pharmacology , Ganciclovir/pharmacology , Genetic Therapy , Glioma/therapy , Interleukin-4/genetics , Simplexvirus/enzymology , Thymidine Kinase/genetics , Viral Proteins/genetics , Animals , Female , Gene Transfer Techniques , Genetic Vectors , Interleukin-4/metabolism , Interleukin-4/therapeutic use , Mice , Mice, Nude , Rats , Rats, Sprague-Dawley , Retroviridae/genetics , Transduction, GeneticABSTRACT
The potential of gene therapy strategies for malignant gliomas that are based on retroviral-mediated transfer of a "suicide gene" such as Herpes Simplex Virus-thymidine kinase HSV-tk and subsequent treatment by a prodrug (ganciclovir, for example), has been emphasized by the promising results obtained by several groups. However, further experimental data as well as preliminary clinical results indicate that the low efficiency of retroviral-mediated gene transfer in vivo as well as difficulties for the diffusion of the prodrug inside the tumour mass can limit the efficacy of this form of gene therapy. To achieve a more effective limitation of tumour growth other approaches may be combined with the "suicide gene" strategy and the enhancement of the immunological response to the tumour by cytokine gene transfer is prominent among these approaches. The authors' experiments in nude mice confirm the antineoplastic role of IL-4 and encourage testing the effects of the simultaneous transfer of IL-4 and HSV-tk genes in immunocompetent animals.
Subject(s)
Cell Death/genetics , Gene Transfer Techniques , Genetic Therapy/methods , Immunotherapy/methods , Animals , Cell Division/genetics , Combined Modality Therapy , Ganciclovir/administration & dosage , Gene Expression Regulation, Neoplastic/physiology , Humans , Interleukin-4/genetics , Mice , Simplexvirus/genetics , Thymidine Kinase/geneticsABSTRACT
The loss of genetic material on chromosome 10q is frequent in different tumors and particularly in malignant gliomas. We analyzed 90 of these tumors and found loss of heterozygosity (LOH) in >90% of the informative loci in glioblastoma multiforme (GBM). Initial studies restricted the common LOH region to 10q24-qter. Subsequently, the study of a pediatric GBM suggested D10S221 and D10S209, respectively, as centromeric and telomeric markers of a 4-cM LOH region. It is interesting to note that, in one subset of cells from this tumor, locus D10S209 seems involved in the allelic imbalance of a larger region, with D10S214 as telomeric marker. This 17-cM region contains the D10S587-D10S216 interval of common deletion recently defined on another set of gliomas.
Subject(s)
Brain Neoplasms/genetics , Chromosomes, Human, Pair 10 , Gene Deletion , Genes, Tumor Suppressor , Glioma/genetics , Brain Neoplasms/pathology , Centromere , Child , Chromosome Mapping , DNA, Neoplasm/analysis , Genetic Markers , Genomic Library , Glioma/pathology , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Magnetic Resonance Imaging , TelomereABSTRACT
We studied the karyotypes of eight differentiated gliomas, 19 anaplastic gliomas, and 23 glioblastomas (GBM). Normal stemlines were present in 70% of the differentiated and anaplastic gliomas; abnormalities were mostly characterized by loss of sex chromosomes. In GBM, on the contrary, only 13% of the stemlines were normal and three groups, 45,XO, near-diploid, and near tetraploid, could be identified. The most frequent alterations among GBM were: total or partial loss of chromosome 10 in nine cases, structural abnormalities of chromosome 9 in seven cases, and loss of the Y chromosome in stemline clones of seven cases. Less frequent abnormalities included chromosomes 7, 1, 3, and 19. Our data support the cytogenetic model of gliomas as multi-stage tumors. GBM, in particular, can originate from the evolution of astrocytomas but can also develop de novo. In both cases loss of genetic material on chromosome 10 seems to play a crucial role.