PubMed Health⌕ Search

Biomedical subjects

Gaetano Romano

Publications and source records attributed to Gaetano Romano.

18 recordsLinked to original sources
PubMed2007

Transcription and epigenetic profile of the promoter, first exon and first intron of the human tyrosine hydroxylase gene.

The transcriptional and chromatin profile of the promoter, first exon and first intron of the human TH gene were analyzed in human neuroblastoma BE(2)-C-16 and human renal carcinoma 293FT cell lines. The latter is a cell culture system that is not permissive for TH gene expression, whereas the former has a 50% cell fraction that tests positive for TH. The engineering of a 6.3 kb recombinant human TH promoter revealed the presence of repressors of transcription between positions (-6,244/-194). The addition of a 1.2 kb fragment of the first intron of the human TH gene (+730/+1,653) enhanced transcriptional activity of the recombinant promoter. However, both constructs were not specific for TH-positive BE(2)-C-16 cells. Chromatin immunoprecipitation (Chip) analysis was carried out on BE(2)-C-16 and 293FT cells to probe sequences of promoter, first exon and first intron of the human TH gene from position (-448/+1,204). The presence of nucleosomes was observed approximately from position (-20/+473) in both cell lines. Chip analysis was then conducted to determine the acetylation of various lysine residues of H3 and H4 in both cell lines. All analyzed lysine residues of H3 and H4 were acetylated in BE(2)-C-16 cells, whereas 293FT cells tested positive for acetylation only in the external lysine residues of the histone tail. Our data are compatible with an active TH gene expression in a 50% cell fraction of BE(2)-C-16 cells. Further analysis of epigenetic programming might lead to the identification of the factors that determine TH gene expression specifically in dopaminergic neurons.

Acetylation↗
PubMed2006

Tyrosine hydroxylase gene regulation in human neuronal progenitor cells does not depend on Nurr1 as in the murine and rat systems.

A previous study on the human tyrosine hydroxylase (TH) promoter revealed remarkable differences in the mechanism of TH gene regulation between the human and murine models. Indeed, a low degree of homology was observed in the sequence of TH promoters among human, mouse, and rat systems. Only five short conserved regions (CRs) could be identified among the three species. A human TH minimal promoter was engineered and assembled into a self-inactivating lentiviral vector system. This human TH minimal promoter contained the five CRs plus the first -194 bp from the transcription start of the human TH promoter and the first 35 bp of the untranslated messenger RNA leader of the human TH gene. A significant degree of specificity for this human TH minimal promoter was observed only for human neuronal progenitor cells (hNPCs), but not for TH-positive differentiated mouse primary striatal and substantia nigra cells, indicating a significant difference in TH gene regulation between the human and mouse systems. Not only is the degree of homology between the human and mouse promoters in the range of only 46%, but also those few elements that share a high degree of homology display totally different functions in human and mouse brain-derived cells. In the rodent system, NR4A2 (Nurr1) is required for the transactivation of TH minimal promoters. Intriguingly, neither the dimeric nor the heterodimeric binding sites for Nurr1 are present in the 13 kb DNA sequence that contains the human TH promoter. Instead, the CRs termed one and four of the human TH promoter encode only for a half palindromic binding site sequence for Nurr1, which failed to bind Nurr1 in an in vitro electrophoretic mobility shift assay (EMSA). Additionally, of the three monomeric NGFI-B response element (NBRE) core sites (AGGTCA) and two NBRE-related sites present in the human TH promoter, only one core and two NBRE-related sites formed protein binding complexes. Interestingly, there was no increase of protein binding complex formation upon TH induction and in no case could antibodies supershift Nurr1 from the complex. These findings, taken together, demonstrate that NBRE-related binding sites for Nurr1 do not play a direct role in mediating an interaction between Nurr1 and the human TH promoter. Likewise, immunohistochemical and Western blot analysis have also confirmed that both endogenous and exogenous Nurr1 expression does not positively correlate with TH gene expression in hNPCs, in contrast to the mouse model. In addition, real-time PCR analysis revealed that the downregulation of human Nurr1 gene expression mediated by silencing RNA molecules did not affect human TH gene expression in differentiated hNPCs. A better understanding of human TH gene regulation may have important implications both for the development of novel therapeutic approaches and the study of the pathogenesis of a variety of neurological illnesses, including Parkinson's disease, bipolar disorder, and schizophrenia.

Animals↗
PubMed2006

The controversial role of adenoviral-derived vectors in gene therapy programs: where do we stand?

The high immunogenicity of recombinant adenoviral vectors is one of the major issues in the field of gene therapy. Adenoviral-based vectors are susceptible both to cytotoxic T-lymphocyte and humoral immune responses. In addition, leaky adenoviral genes also render transduced cells susceptible to host immune responses. These are the main reasons why adenoviral-based vectors are not suitable to correct genetic disorders, which require long-term expression of the transgene. Another limit to long-term transgene expression is posed by the fact that adenoviral-based vectors do not integrate their genome into the cellular chromosomal DNA of transduced cell populations. As it stands, adenoviral-mediated gene transfer is a promising tool for cancer therapy and for genetic immunization programs against infectious diseases, provided that host immune responses are carefully controlled.

Adenoviridae↗
PubMed2006

Perspectives and controversies in the field of stem cell research.

The fourth annual meeting of the International Society for Stem Cell Research focused on a number of pressing issues, including: (I) the need to better characterize the biology of stem cells; (II) the need to exploit and optimize the great therapeutic potential of stem cells in tissue regeneration; (III) ethical and safety considerations related to the use of human embryonic stem cells; (IV) the contribution of adult stem cells to carcinogenesis; (V) the need to investigate the biology of cancer stem cells. The purpose of this report is to summarize the current status of stem cell research, as surmised by the proceedings of this meeting.

Adult↗
PubMed2005

Characterization of five evolutionary conserved regions of the human tyrosine hydroxylase (TH) promoter: implications for the engineering of a human TH minimal promoter assembled in a self-inactivating lentiviral vector system.

A DNA fragment of about 13 kb containing the human tyrosine hydroxylase (TH) promoter was previously isolated from a genomic DNA library and sequenced. The 11 kb from the transcription start of the human TH promoter was successively joined to the green fluorescent protein (GFP) to generate a transgenic mouse model. High levels of GFP expression could be observed in TH-positive cells of the Substantia nigra of embryonic and adult mice. Intriguingly, the sequence of the human TH promoter showed a low degree of homology with the mouse and rat TH promoters. In fact, comparative analysis of the sequences of human, rat, and mouse TH promoters revealed only five small regions of high homology. These five evolutionarily conserved regions were numbered in numeric progression from the 5' end of human TH promoter. In the present study, a panel of minimal human TH promoters was generated to analyze the transcriptional activity and specificity of gene expression conferred by the five conserved regions (CRs). The series of constructs was termed 250 bp and contained the first -194 bp of the human TH promoter immediately upstream of the transcription start, the first 35 bp the human TH messenger RNA leader, plus one or more of the five CRs. All the constructs were assembled in a self-inactivating form of the latest series of lentiviral vector system based on the human immunodeficiency virus type 1 (HIV-1). Lentiviral-mediated gene transfer was highly efficient for the in vitro transduction of human neuronal progenitor cells (hNPCs). Since a subset of hNPCs express TH following in vitro treatment with a mixture of differentiating agents, it was possible to assess specificity of expression for all the minimal human TH promoters. Overall, the successive addition of the five conserved regions produced a greater degree of specificity in induced TH-positive hNPCs, in particular after the addition of CRI (-8,917, -8,876). However, the human TH minimal promoters did not show any specificity for TH-positive differentiated mouse primary striatal and S. nigra cells, indicating a difference of TH gene regulation between the human and mouse systems. The human TH minimal promoters may provide the opportunity for the selection of TH-positive human embryonic and adult stem cells for brain transplantation experiments in animal models for Parkinson's disease.

Animals↗
PubMed2005

Current development of lentiviral-mediated gene transfer.

Lentiviral-based vectors have been widely used lately in preclinical studies and for the in vitro genetic manipulation of embryonic and adult stem cells. They allow for the transduction of nondividing cells and for stable gene expression. On these grounds, lentiviral vectors look promising for eventual applications for the gene-based treatment of neurological disorders, cardiopathies and inherited or acquired genetic diseases. The best developed and characterized lentiviral vector system is based on the human immunodeficiency virus type 1 (HIV-1). However, safety concerns preclude any possible application of HIV-1-derived vectors in clinical trials. For this reason, gene therapists are currently improving the design of other lentiviral vectors, such as feline immunodeficiency virus, equine anemia infectious virus and Visna virus. These lentiviruses are not pathogenic in humans and are only distantly related to primate Retroviridae. This review summarizes the achievements in improving the design of lentiviral vector systems that are not based on HIV-1.

Animals↗
PubMed2005

The role of adult stem cells in carcinogenesis.

The role of adult stem cells in tissue regeneration has attracted much interest because of its enormous therapeutic potential. Adult stem cells reside in every tissue of the body and have the ability to self-renew and to give rise to a high number of differentiated progeny. These are indeed the essential steps in tissue regeneration, which in some cases occurs constitutively, whereas in other cases happens in response to an injury. The identification of the key factors involved in self-renewal and differentiation pathways is at the top of the agenda of stem cell biologists. Interestingly, a number of factors that govern the fate of adult stem cells also play a role in malignant cell transformation, such as the Wnt cascade, Oct-4, Bmi-1 and Evi1. In addition, rare subpopulations of cancer stem cells were identified for leukemia and other solid tumors. These rare fractions of cancer stem cells are in large part responsible for maintaining the tumor mass, as they have the capacity to self-renew and to generate a high number of progeny via differentiation program. These findings can be considered a major breakthrough in cancer research. Currently, oncologists and stem cell biologists have two main goals: i) the assessment of the consistency of the line that divides adult stem cells and carcinogenesis; ii) to better characterize the biology of cancer stem cells. This new line of investigation may allow for the development of novel therapeutics and cancer diagnostics.

AC133 Antigen↗
PubMed2004

pRb2/p130 decreases sensitivity to apoptosis induced by camptothecin and doxorubicin but not by taxol.

PURPOSE: In addition to their original function as cell cycle regulators, retinoblastoma (Rb) family members were recently reported to modulate the sensitivity of cancer cells to chemotherapeutic agents. The purpose of this study is to investigate the possible role of pRb2/p130 in the sensitivity of ovarian cancer to camptothecin, doxorubicin, and taxol. EXPERIMENTAL DESIGN: pRb2/p130 was overexpressed in the CAOV-3 ovarian cancer cell line, and the effect of pRb2/p130 overexpression on sensitivity to apoptosis trigged by IC(50) doses of different drugs was evaluated by various methods, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and Western blot analyses. RESULTS: The results reported in this study support the conclusion that overexpression of pRb2/p130 in the CAOV-3 ovarian cancer cell line lacking wild-type p53 is able to inhibit apoptosis triggered by camptothecin and doxorubicin through the c-Jun NH(2)-terminal kinase signaling transduction pathway. Conversely, taxol-induced cell death is not influenced by the pRb2/p130 protein level. CONCLUSIONS: A careful analysis of pRb2/p130 expression in tumor specimens could help to identify the best clinical protocol to be used for each patient, improving efficacy and tolerance and therefore offering additional progress in the treatment of advanced ovarian cancer.

Adenoviridae↗
PubMed2004

Stem cell transplantation therapy: controversy over ethical issues and clinical relevance.

The possibility of regenerating tissues would provide an effective therapeutic tool for the treatment of many pathological conditions, including neurological diseases, spinal cord injuries, cardiopathies, diabetes, hematological illnesses and genetic disorders. While stem cells may have the potential of regenerating a variety of tissues, as indicated by a number of groundbreaking but preliminary reports, ethical issues and safety considerations seem to preclude the use of human embryonic stem cells in the clinical setting. Adult stem cells might circumvent the issues posed by embryonic stem cells, although the potential plasticity of adult stem cells is under scrutiny because of many conflicting and contradictory reports in the field of stem cell research. Indeed, many aspects of the biology of stem cells are still not known. In this respect, stem cell biologists have to address several pressing issues. A better understanding of stem cell biology would almost certainly allow for the establishment of efficient and reliable cell transplantation experimental programs in the clinic.

Cell Differentiation↗
PubMed2004

Systems for regulated or tissue-specific gene expression.

The achievement of efficient regulation and/or tissue-specific gene expression is one of the major goals for gene therapists. A number of inducible systems and tissue-specific promoters are currently available. Studies in vitro and in vivo have shown promising results in controlling gene expression. However, the achievement of efficient gene regulation in patients is still an open question. This review describes the main inducible systems and tissue-specific promoters in view of their potential therapeutic applications.

Animals↗
PubMed2003

Gene transfer in experimental medicine.

Gene transfer technology has many potential applications in medicine. Phase I and phase II gene-based clinical trials have been conducted for the treatment of cancer, monogenic disorders, some neurodegenerative illnesses, cardiopathies and infectious diseases. A phase I gene therapy clinical trial has recently been approved for the treatment of Parkinson's disease, while preclinical studies are in progress to develop gene-based interventions for the treatment of Alzheimer's disease and Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury, and diabetes type 1 and type 2. A number of gene transfer models have been generated for gene therapy and genetic immunization programs. Vector design is addressing several pressing issues in the matter of gene delivery improvement, stabilization of transgene expression and safety. This is necessary in order to achieve efficient gene-based therapeutic interventions. Indeed, considerable progress has been reported in the field of vector design, which has produced some encouraging results in clinical trials and preclinical studies. However, vector design should be further developed to allow for the successful application of gene transfer technology in therapy. This review summarizes the latest achievements and controversies in clinical trials and preclinical studies in the field of gene therapy.

Animals↗
PubMed2003

The complex biology of the receptor for the insulin-like growth factor-1.

The receptor for the insulin-like growth factor-1 (IGF-1R) controls a wide variety of cellular functions. IGF-1R is a tyrosine kinase receptor that is activated by the binding of a ligand to the extracellular domain. There are three ligands for IGF-1R: IGF-I, IGF-II and insulin at supraphysiological concentrations. The binding of the ligand activates the catalytic activity of the tyrosine kinase domain, which in turn causes the autophosphorylation of IGF-1R. As a consequence of autophosphorylation, the activated IGF-1R sends a potent mitogenic signal to the cell nucleus. It has been clearly shown that an overexpressed and activated IGF-1R is quasi-obligatory for the establishment of a malignant cell phenotype. Interestingly, the targeting of IGF-1R can reverse the malignant phenotype in cancer cells, without affecting the biology of normal cells. For these reasons, IGF-1R seems to be a very promising candidate target for cancer therapy. In addition, IGF-1R protects cells from apoptosis and promotes cell growth and proliferation. However, more recent studies have also shown that IGF-1R regulates cell adhesion and motility, and, in certain cellular contexts, can induce differentiation. Indeed, the biology of the IGF-1R appears more complex than previously thought. The goal of this review is to describe the multiple functions displayed by IGF-1R in cell biology.

Animals↗
PubMed2002

Macroamylasemia in a patient with multiple myeloma.

We report a rare case of a patient with multiple myeloma who developed hyperamylasemia not associated to hyperamylasuria and without symptoms of pancreatic or salivary disease. This condition suggested the occurrence of macroamylasemia, consisting of macromolecules of amylase bound with immunoglobulins, which are not filtered by the kidneys. Hyperamylasemia was not present at the diagnosis of myeloma and appeared at the relapse of the disease, simultaneously with the appearance of an additional gamma-chain oligoclonal component, suggesting a possible role of these chains in producing macroamylasemia. To our knowledge, this is the first report of macroamylasemia in a patient with multiple myeloma.

Amylases↗
PubMedDate not supplied

Human immunodeficiency virus type 1 (HIV-1) derived vectors: safety considerations and controversy over therapeutic applications.

The latest generation of lentiviral vectors based on HIV-1 is one of the most efficient tools for gene transduction of mammalian cells. However, the possible employment of HIV-based vectors in clinical trials is a very controversial issue, mainly due to safety and ethical concerns. HIV-1 is a lethal pathogenic agent, which induces AIDS. Genetic vectors must derive either from viruses that are not pathogenic in humans, or that eventually just cause mild illnesses. Patients exposed to HIV-based vectors will test seropositive to certain components of HIV-1. In addition, there might be other possible adverse effects in patients that cannot be predicted, as many aspects of the pathogenesis of AIDS have not been completely understood yet. On these grounds, it seems necessary to improve the design of other lentiviral vectors, which derive from viruses that are not pathogenic in humans and are distantly related to primate retroviridae.

Genetic Therapy↗
PubMedDate not supplied

Advances and perspectives in the field of gene transfer technology.

The American Society of Gene Therapy 9th annual meeting, held May 31 to June 4, 2006, in Baltimore (Maryland, U.S.A.), focused on several aspects of gene transfer technology, such as vector development of a wide variety of gene delivery systems, stem cell research, gene expression, engineering of cell and animal models for the study of several pathological conditions, genetic immunization procedures, immunogenicity of vectors and transgenes, insertional mutagenesis and last, but not least, clinical and preclinical studies. Some advances were reported in the field of gene transfer technology; however, safety issues, such as insertional mutagenesis, and improvement of vector design remain at the top of the agenda of gene therapists.

Animals↗