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1.
Radiat Res ; 197(1): 43-56, 2022 01 01.
Article in English | MEDLINE | ID: mdl-33857285

ABSTRACT

Experimental mouse studies are important to gain a comprehensive, quantitative and mechanistic understanding of the biological factors that modify individual risk of radiation-induced health effects, including age at exposure, dose, dose rate, organ/tissue specificity and genetic factors. In this study, neonatal Ptch1+/- mice bred on CD1 and C57Bl/6 background received whole-body irradiation at postnatal day 2. This time point represents a critical phase in the development of the eye lens, cerebellum and dentate gyrus (DG), when they are also particularly susceptible to radiation effects. Irradiation was performed with γ rays (60Co) at doses of 0.5, 1 and 2 Gy, delivered at 0.3 Gy/min or 0.063 Gy/min. Wild-type and mutant mice were monitored for survival, lens opacity, medulloblastoma (MB) and neurogenesis defects. We identified an inverse genetic background-driven relationship between the radiosensitivity to induction of lens opacity and MB and that to neurogenesis deficit in Ptch1+/- mutants. In fact, high incidence of radiation-induced cataract and MB were observed in Ptch1+/-/CD1 mutants that instead showed no consequence of radiation exposure on neurogenesis. On the contrary, no induction of radiogenic cataract and MB was reported in Ptch1+/-/C57Bl/6 mice that were instead susceptible to induction of neurogenesis defects. Compared to Ptch1+/-/CD1, the cerebellum of Ptch1+/-/C57Bl/6 mice showed increased radiosensitivity to apoptosis, suggesting that differences in processing radiation-induced DNA damage may underlie the opposite strain-related radiosensitivity to cancer and non-cancer pathologies. Altogether, our results showed lack of dose-rate-related effects and marked influence of genetic background on the radiosensitivity of Ptch1+/-mice, supporting a major contribution of individual sensitivity to radiation risk in the population.


Subject(s)
Medulloblastoma/ethnology , Neoplasms, Radiation-Induced/etiology , Animals , Dose-Response Relationship, Radiation , Gamma Rays , Genetic Background , Humans , Lens, Crystalline/radiation effects , Mice, Inbred C57BL , Neurogenesis , Radiation Tolerance , Whole-Body Irradiation
2.
Radiat Res ; 197(1): 22-35, 2022 01 01.
Article in English | MEDLINE | ID: mdl-33857324

ABSTRACT

One harmful long-term effect of ionizing radiation is cataract development. Recent studies have been focused on elucidating the mechanistic pathways involved in this pathogenesis. Since accumulating evidence has established a role of microRNAs in ocular diseases, including cataract, the goal of this work was to determine the microRNA signature of the mouse lens, at short time periods postirradiation, to understand the mechanisms related to radio-induced cataractogenesis. To evaluate the differences in the microRNA profiles, 10-week-old Patched1 heterozygous (Ptch1+/-) mice, bred onto two different genetic backgrounds (CD1 and C57Bl/6J), received whole-body 2 Gy γ-ray irradiation, and 24 h later lenses were collected. Next-generation sequencing and bioinformatics analysis revealed that genetic background markedly influenced the list of the deregulated microRNAs and the mainly predicted perturbed biological functions of 2 Gy irradiated Ptch1+/- mouse lenses. We identified a subset of microRNAs with a contra-regulated expression between strains, with a key role in regulating Toll-like receptor (TLR)-signaling pathways. Furthermore, a detailed analysis of miRNome data showed a completely different DNA damage response in mouse lenses 24 h postirradiation, mainly mediated by a marked upregulation of p53 signaling in Ptch1+/-/C57Bl/6J lenses that was not detected on a CD1 background. We propose a strict interplay between p53 and TLR signaling in Ptch1+/-/C57Bl/6J lenses shortly after irradiation that could explain both the resistance of this strain to developing lens opacities and the susceptibility of CD1 background to radiation-induced cataractogenesis through activation of epithelial-mesenchymal transition.


Subject(s)
Cataract/etiology , Lens, Crystalline/radiation effects , Animals , DNA Damage/radiation effects , Disease Models, Animal , Dose-Response Relationship, Radiation , Gamma Rays , Genetic Background , Humans , Mice, Inbred C57BL , MicroRNAs , Patched-1 Receptor/metabolism , Signal Transduction , Toll-Like Receptors/metabolism , Whole-Body Irradiation
3.
J Exp Clin Cancer Res ; 38(1): 311, 2019 Jul 16.
Article in English | MEDLINE | ID: mdl-31311580

ABSTRACT

BACKGROUND: Glioblastoma is the most aggressive and most lethal primary brain tumor in the adulthood. Current standard therapies are not curative and novel therapeutic options are urgently required. Present knowledge suggests that the continued glioblastoma growth and recurrence is determined by glioblastoma stem-like cells (GSCs), which display self-renewal, tumorigenic potential, and increased radio- and chemo-resistance. The G-quadruplex ligand RHPS4 displays in vitro radiosensitizing effect in GBM radioresistant cells through the targeting and dysfunctionalization of telomeres but RHPS4 and Ionizing Radiation (IR) combined treatment efficacy in vivo has not been explored so far. METHODS: RHPS4 and IR combined effects were tested in vivo in a heterotopic mice xenograft model and in vitro in stem-like cells derived from U251MG and from four GBM patients. Cell growth assays, cytogenetic analysis, immunoblotting, gene expression and cytofluorimetric analysis were performed in order to characterize the response of differentiated and stem-like cells to RHPS4 and IR in single and combined treatments. RESULTS: RHPS4 administration and IR exposure is very effective in blocking tumor growth in vivo up to 65 days. The tumor volume reduction and the long-term tumor control suggested the targeting of the stem cell compartment. Interestingly, RHPS4 treatment was able to strongly reduce cell proliferation in GSCs but, unexpectedly, did not synergize with IR. Lack of radiosensitization was supported by the GSCs telomeric-resistance observed as the total absence of telomere-involving chromosomal aberrations. Remarkably, RHPS4 treatment determined a strong reduction of CHK1 and RAD51 proteins and transcript levels suggesting that the inhibition of GSCs growth is determined by the impairment of the replication stress (RS) response and DNA repair. CONCLUSIONS: We propose that the potent antiproliferative effect of RHPS4 in GSCs is not determined by telomeric dysfunction but is achieved by the induction of RS and by the concomitant depletion of CHK1 and RAD51, leading to DNA damage and cell death. These data open to novel therapeutic options for the targeting of GSCs, indicating that the combined inhibition of cell-cycle checkpoints and DNA repair proteins provides the most effective means to overcome resistance of GSC to genotoxic insults.


Subject(s)
Acridines/administration & dosage , Brain Neoplasms/therapy , Glioblastoma/therapy , Neoplastic Stem Cells/drug effects , Radiation-Sensitizing Agents/administration & dosage , Acridines/pharmacology , Animals , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Cell Survival/drug effects , Cell Survival/radiation effects , Checkpoint Kinase 1/genetics , Checkpoint Kinase 1/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Glioblastoma/genetics , Glioblastoma/metabolism , Humans , Mice , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Radiation-Sensitizing Agents/pharmacology , Xenograft Model Antitumor Assays
4.
DNA Repair (Amst) ; 74: 70-79, 2019 02.
Article in English | MEDLINE | ID: mdl-30606609

ABSTRACT

DSBs are harmful lesions produced through endogenous metabolism or by exogenous agents such as ionizing radiation, that can trigger genomic rearrangements. We have recently shown that exposure to 2 Gy of X-rays has opposite effects on the induction of Shh-dependent MB in NHEJ- and HR-deficient Ptch1+/- mice. In the current study we provide a comprehensive link on the role of HR/NHEJ at low doses (0.042 and 0.25 Gy) from the early molecular changes through DNA damage processing, up to the late consequences of their inactivation on tumorigenesis. Our data indicate a prominent role for HR in genome stability, by preventing spontaneous and radiation-induced oncogenic damage in neural precursors of the cerebellum, the cell of origin of MB. Instead, loss of DNA-PKcs function increased DSBs and apoptosis in neural precursors of the developing cerebellum, leading to killing of tumor initiating cells, and suppression of MB tumorigenesis in DNA-PKcs-/-/Ptch1+/- mice. Pathway analysis demonstrates that DNA-PKcs genetic inactivation confers a remarkable radiation hypersensitivity, as even extremely low radiation doses may deregulate many DDR genes, also triggering p53 pathway activation and cell cycle arrest. Finally, by showing that DNA-PKcs inhibition by NU7441 radiosensitizes human MB cells, our in vitro findings suggest the inclusion of MB in the list of tumors beneficiating from the combination of radiotherapy and DNA-PKcs targeting, holding promise for clinical translation.


Subject(s)
Cerebellar Neoplasms/genetics , DNA Repair/radiation effects , Medulloblastoma/genetics , Neoplasms, Radiation-Induced/genetics , Patched-1 Receptor/deficiency , Patched-1 Receptor/metabolism , Animals , Carcinogenesis/genetics , Carcinogenesis/radiation effects , Cell Line, Tumor , Cerebellar Neoplasms/metabolism , Cerebellar Neoplasms/pathology , Cerebellar Neoplasms/therapy , DNA Damage , DNA End-Joining Repair/radiation effects , DNA Helicases/genetics , DNA-Activated Protein Kinase/deficiency , DNA-Binding Proteins/deficiency , Dose-Response Relationship, Radiation , Homologous Recombination/radiation effects , Humans , Medulloblastoma/metabolism , Medulloblastoma/pathology , Medulloblastoma/therapy , Mice , Molecular Targeted Therapy , Mutation , Neoplasms, Radiation-Induced/metabolism , Neoplasms, Radiation-Induced/pathology , Neoplasms, Radiation-Induced/therapy , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Risk , X-Rays/adverse effects
5.
Curr Mol Med ; 12(5): 613-24, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22452594

ABSTRACT

A long-held dogma in radiation biology has been that the biological effects of exposure to ionizing radiation occur as a result of damage in directly irradiated cells and that no effect would occur in neighboring unirradiated cells. This paradigm has been frequently challenged by reports of radiation effects in unirradiated or 'bystander' cells receiving signals from directly irradiated cells, an issue that may have substantial impact on radiation risk assessment and development of radiation-based therapies. Radiation-induced bystander effects have been shown in single-cell systems in vitro for an array of cancer relevant endpoints, and may trigger damage in more complex 3-D tissue systems. They may be mediated by soluble factors released by irradiated cells into the extracellular environment and/or by the passage of mediator molecules through gap-junction intercellular communication. To date, evidence that radiation-associated bystander or abscopal responses are effectual in vivo has been limited, but new data suggest that they may significantly affect tumor development in susceptible mouse models. Further understanding of how the signal/s is transmitted to unirradiated cells and tissues and how it provokes long-range and significant responses is crucial. By summarizing the existing evidence of radiation induced bystander-like effects in various systems with emphasis on in vivo findings, we will discuss the potential mechanisms involved in these observations and how effects in bystander cells contribute to uncertainties in assessing cancer risks associated with radiation exposure.


Subject(s)
Bystander Effect/physiology , Radiation, Ionizing , Animals , Cell Communication , DNA Damage/radiation effects , Humans , Neoplasms/etiology
6.
Oncogene ; 30(45): 4601-8, 2011 Nov 10.
Article in English | MEDLINE | ID: mdl-21602884

ABSTRACT

Ionizing radiation is a genotoxic agent and human carcinogen. Recent work has questioned long-held dogmas by showing that cancer-associated genetic alterations occur in cells and tissues not directly exposed to radiation, questioning the robustness of the current system of radiation risk assessment. In vitro, diverse mechanisms involving secreted soluble factors, gap junction intercellular communication (GJIC) and oxidative metabolism are proposed to mediate these indirect effects. In vivo, the mechanisms behind long-range 'bystander' responses remain largely unknown. Here, we investigate the role of GJIC in propagating radiation stress signals in vivo, and in mediating radiation-associated bystander tumorigenesis in mouse central nervous system using a mouse model in which intercellular communication is downregulated by targeted deletion of the connexin43 (Cx43) gene. We show that GJIC is critical for transmission of oncogenic radiation damage to the non-targeted cerebellum, and that a mechanism involving adenosine triphosphate release and upregulation of Cx43, the major GJIC constituent, regulates transduction of oncogenic damage to unirradiated tissues in vivo. Our data provide a novel hypothesis for transduction of distant bystander effects and suggest that the highly branched nervous system, similar to the vascular network, has an important role.


Subject(s)
Adenosine Triphosphate/metabolism , Bystander Effect/radiation effects , Cell Transformation, Neoplastic/genetics , Cerebellar Neoplasms/genetics , Connexin 43/metabolism , DNA Damage/genetics , Neoplasms, Radiation-Induced/genetics , Animals , Cerebellum/metabolism , Cerebellum/radiation effects , Connexin 43/genetics , Gap Junctions/metabolism , Gap Junctions/radiation effects , Mice , Radiation Dosage , Sequence Deletion/radiation effects , Signal Transduction/radiation effects
7.
Oncogene ; 30(47): 4740-9, 2011 Nov 24.
Article in English | MEDLINE | ID: mdl-21602895

ABSTRACT

Heterozygous Patched1 (Ptc1(+/-)) mice are prone to medulloblastoma (MB), and exposure of newborn mice to ionizing radiation dramatically increases the frequency and shortens the latency of MB. In Ptc1(+/-) mice, MB is characterized by loss of the normal remaining Ptc1 allele, suggesting that genome rearrangements may be key events in MB development. Recent evidence indicates that brain tumors may be linked to defects in DNA-damage repair processes, as various combinations of targeted deletions in genes controlling cell-cycle checkpoints, apoptosis and DNA repair result in MB in mice. Non-homologous end joining (NHEJ) and homologous recombination (HR) contribute to genome stability, and deficiencies in either pathway predispose to genome rearrangements. To test the role of defective HR or NHEJ in tumorigenesis, control and irradiated Ptc1(+/-) mice with two, one or no functional Rad54 or DNA-protein kinase catalytic subunit (DNA-PKcs) alleles were monitored for MB development. We also examined the effect of Rad54 or DNA-PKcs deletion on the processing of endogenous and radiation-induced double-strand breaks (DSBs) in neural precursors of the developing cerebellum, the cells of origin of MB. We found that, although HR and NHEJ collaborate in protecting cells from DNA damage and apoptosis, they have opposite roles in MB tumorigenesis. In fact, although Rad54 deficiency increased both spontaneous and radiation-induced MB development, DNA-PKcs disruption suppressed MB tumorigenesis. Together, our data provide the first evidence that Rad54-mediated HR in vivo is important for suppressing tumorigenesis by maintaining genomic stability.


Subject(s)
Cerebellar Neoplasms/etiology , DNA End-Joining Repair , Homologous Recombination , Medulloblastoma/etiology , Receptors, Cell Surface/physiology , Animals , Cerebellar Neoplasms/genetics , DNA Damage , DNA Helicases/physiology , DNA-Activated Protein Kinase/physiology , Genomic Instability , Loss of Heterozygosity , Medulloblastoma/genetics , Mice , Nuclear Proteins/physiology , Patched Receptors , Patched-1 Receptor , Receptors, Cell Surface/genetics , Risk
8.
Carcinogenesis ; 30(2): 340-7, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18952596

ABSTRACT

Patched1 heterozygous mice (Ptch1(+/-)) are useful for basal cell carcinoma (BCC) studies, being remarkably susceptible to BCC induction by ultraviolet or ionizing radiation. Analogously, skin carcinogenesis-susceptible (Car-S) mice are elective for studies of papilloma and squamous cell carcinoma (SCC) induction. We previously reported a striking effect of gender on BCC induction in Ptch1(+/-) mice, with total resistance of females; likewise, Car-S females show increased skin tumor resistance relative to males. Here, we investigated the protective role of endogenous estrogen in skin keratinocyte tumorigenesis. Control (CN) and ovariectomized Ptch1(+/-) or Car-S females were irradiated for BCC induction or topically treated with chemical carcinogens for SCC induction. Susceptibility to BCC or SCC was dramatically increased in ovariectomized Ptch1(+/-) and Car-S females and restored to levels observed in males. Remarkably, progression of initially benign papillomas to malignant SCC occurred only in ovariectomized Car-S females. We explored the mechanisms underlying tumor progression and report overexpression of estrogen receptor (ER)-alpha, downregulation of ERbeta and upregulation of cyclin D1 in papillomas from ovariectomized Car-S relative to papillomas from CN females. Thus, an imbalanced ERalpha/ERbeta expression may be associated with estrogen-mediated modulation of non-melanoma skin carcinogenesis, with a key role played by cyclin D1. Our findings underscore a highly protective role of endogenous estrogen against skin tumorigenesis by diverse agents in two independent mouse models of skin cancer.


Subject(s)
Carcinoma, Basal Cell/metabolism , Carcinoma, Squamous Cell/metabolism , Estrogens/physiology , Skin Neoplasms/metabolism , Animals , Carcinoma, Basal Cell/pathology , Carcinoma, Squamous Cell/pathology , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cyclin D1/metabolism , Disease Models, Animal , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/metabolism , Female , Male , Mice , Neoplasms, Radiation-Induced/metabolism , Neoplasms, Radiation-Induced/pathology , Ovariectomy , Papilloma/metabolism , Papilloma/pathology , Patched Receptors , Patched-1 Receptor , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Skin Neoplasms/pathology , Ultraviolet Rays
9.
Radiat Res ; 168(6): 733-40, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18088186

ABSTRACT

Patched1 heterozygous knockout mice (Ptc1+/-), an animal model of multiorgan tumorigenesis in which ionizing radiation dramatically accelerates tumor development, were used to study the potential tumorigenic effects of electromagnetic fields (EMFs) on neonatal mice. Two hundred Ptc1+/- mice and their wild-type siblings were enrolled in this study. Newborn mice were exposed to 900 MHz radiofrequency radiation (average SAR: 0.4 W/kg for 5 days, 0.5 h twice a day) or were sham exposed. We found that RF EMFs simulating the Global System for Mobile Communications (GSM) did not affect the survival of the mice, because no statistically significant differences in survival were found between exposed and sham-exposed animals. Also, no effects attributable to radiofrequency radiation were observed on the incidence and histology of Ptc1-associated cerebellar tumors. Moreover, the skin phenotype was analyzed to look for proliferative effects of RF EMFs on the epidermal basal layer and for acceleration of preneoplastic lesions typical of the basal cell carcinoma phenotype of this model. We found no evidence of proliferative or promotional effects in the skin from neonatal exposure to radiofrequency radiation. Furthermore, no difference in Ptc1-associated rhabdomyosarcomas was detected between sham-exposed and exposed mice. Thus, under the experimental conditions tested, there was no evidence of life shortening or tumorigenic effects of neonatal exposure to GSM RF radiation in a highly tumor-susceptible mouse model.


Subject(s)
Heterozygote , Radio Waves , Receptors, Cell Surface/metabolism , Animals , Animals, Newborn , Disease-Free Survival , Electromagnetic Fields , Mice , Mice, Transgenic , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Neoplasms/radiotherapy , Patched Receptors , Patched-1 Receptor , Precancerous Conditions/genetics , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , Precancerous Conditions/radiotherapy , Receptors, Cell Surface/genetics , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Skin Neoplasms/radiotherapy
10.
Oncogene ; 25(40): 5575-80, 2006 Sep 07.
Article in English | MEDLINE | ID: mdl-16636673

ABSTRACT

Inactivation of one Ptc1 allele predisposes humans and mice to spontaneous medulloblastoma development, and irradiation of newborn Ptc1 heterozygous mice results in dramatic increase of medulloblastoma incidence. While a role for loss of wild-type (wt) Ptc1 (LOH) in radiation-induced medulloblastomas from Ptc1(neo67/+) mice is well established, the importance of this event in spontaneous medulloblastomas is still unclear. Here, we demonstrate that biallelic Ptc1 loss plays a crucial role in spontaneous medulloblastomas, as shown by high rate of wt Ptc1 loss in spontaneous tumors. In addition, remarkable differences in chromosomal events involving the Ptc1 locus in spontaneous and radiation-induced medulloblastomas suggest distinct mechanisms for Ptc1 loss. To assess when, during tumorigenesis, Ptc1 loss occurs, we characterized cerebellar abnormalities that precede tumor appearance in Ptc1(neo67/+) mice. We show that inactivation of only one copy of Ptc1 is sufficient to give rise to abnormal cerebellar proliferations with different degree of altered cell morphology, but lacking potential to progress to neoplasia. Furthermore, we identify biallelic Ptc1 loss as the event causally related to the transition from the preneoplastic stage to full blown medulloblastoma. These results underscore the utility of the Ptc1(neo67/+) mouse model for studies on the mechanisms of medulloblastoma and for development of new therapeutic strategies.


Subject(s)
Cerebellar Neoplasms/genetics , Medulloblastoma/genetics , Precancerous Conditions/genetics , Receptors, Cell Surface/genetics , Aging , Animals , Cerebellar Neoplasms/pathology , Chromosomes, Mammalian , Disease Models, Animal , Disease Progression , Hedgehog Proteins , Loss of Heterozygosity , Medulloblastoma/pathology , Mice , Mice, Inbred C57BL , Patched Receptors , Patched-1 Receptor , Precancerous Conditions/pathology , Radiation, Ionizing , Signal Transduction , Trans-Activators/metabolism
11.
Oncogene ; 25(8): 1165-73, 2006 Feb 23.
Article in English | MEDLINE | ID: mdl-16407852

ABSTRACT

Hemizygous Ptc1 mice have many features of Gorlin syndrome, including predisposition to medulloblastoma development. Ionizing radiation synergize with Ptc1 mutation to induce medulloblastoma only in neonatally exposed mice. To explore the mechanisms underlying age-dependent susceptibility, we irradiated Ptc(neo67/+) mice at postnatal day 1 (P1) or 10 (P10). We observed a dramatic difference in medulloblastoma incidence, which ranged from 81% in the cerebellum irradiated at P1 to 3% in the cerebellum irradiated at P10. A striking difference was also detected in the frequency of cerebellar preneoplastic lesions (100 versus 14%). Our data also show significantly lower induction of apoptosis in the cerebellum of medulloblastoma-susceptible (P1) compared to -resistant (P10) mice, strongly suggesting that medulloblastoma formation in Ptc1 mutants may be associated with resistance to radiation-induced cell killing. Furthermore, in marked contrast with P10 mice, cerebellum at P1 displays substantially increased activation of the cell survival-promoting Akt/Pkb protein, and markedly decreased p53 levels in response to radiation-induced genotoxic stress. Overall, these results show that developing cerebellar granule neuron precursors' (CGNPs) radiosensitivity to radiation-induced cell death increases with progressing development and inversely correlates with their ability to neoplastically transform.


Subject(s)
Cell Transformation, Neoplastic/radiation effects , Cerebellar Neoplasms/etiology , DNA Damage/radiation effects , Heterozygote , Medulloblastoma/etiology , Neoplasms, Radiation-Induced/genetics , Allelic Imbalance , Animals , Animals, Newborn , Apoptosis/radiation effects , Cerebellar Neoplasms/pathology , Cerebellum/radiation effects , Incidence , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , Medulloblastoma/pathology , Membrane Proteins/genetics , Membrane Proteins/physiology , Mice , Mice, Knockout , Patched Receptors , Patched-1 Receptor , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Cell Surface , Tumor Suppressor Protein p53/metabolism , X-Rays
12.
Toxicol In Vitro ; 15(4-5): 289-95, 2001.
Article in English | MEDLINE | ID: mdl-11566551

ABSTRACT

The involvement of the NO pathway in several intestinal inflammatory diseases is under investigation. In vitro models may provide a useful approach to better characterise this pathway at the cellular level. For this purpose, we have used Caco-2 cells, which are able to spontaneously differentiate in long-term culture to small intestine enterocytes. The effect of different NO pathway inducers [gamma-interferon (IFN-gamma) and phorbol myristate acetate (PMA)] has been studied. Our results demonstrate that Caco-2 cells constitutively express NOS at very low levels, while the induction with PMA+IFN-gamma triggers the expression of the inducible isoform with a stronger effect starting from day 14 of differentiation. The use of specific inhibitors of gene expression, at transcriptional and translational level, suggests that new synthesis of iNOS mRNA is required, through direct activation of the gene or new synthesis of transcription-required factors, as indicated by CHX inhibition. The morphological alteration induced by PMA+IFN-gamma is reversed by iNOS inhibitor, suggesting that the NO pathway may be involved in the cytoskeletal alterations. The DSP toxins, OA and DTX-1, induce NO production at levels corresponding to their different toxicity, previously detected in Caco-2 cells.


Subject(s)
Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Enzyme Inhibitors/pharmacology , Marine Toxins/pharmacology , Nitric Oxide/biosynthesis , Blotting, Western , Caco-2 Cells/pathology , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Combinations , Enzyme Induction/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Humans , Interferon-gamma/pharmacology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Okadaic Acid/pharmacology , Pyrans/pharmacology , RNA, Messenger/metabolism , Tetradecanoylphorbol Acetate/pharmacology , omega-N-Methylarginine/pharmacology
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