PubMed Health⌕ Search

Biomedical subjects

P P Pandolfi

Publications and source records attributed to P P Pandolfi.

At least 19 recordsLinked to original sources
PubMed1996

Interferon augments PML and PML/RAR alpha expression in normal myeloid and acute promyelocytic cells and cooperates with all-trans retinoic acid to induce maturation of a retinoid-resistant promyelocytic cell line.

The PML gene is fused to the retinoic acid receptor alpha gene (RAR alpha) in the acute promyelocytic leukemia (APL) 15; 17 translocation. PML is expressed in diverse tissues and cell lines and localized in the nucleus with a typical speckled pattern. In the bone marrow, it is preferentially expressed in myeloid cells. PML appears to be transcriptionally regulated by class I and II interferons, which raises the possibility that interferons modulate the function and growth and differentiation potential of normal myeloid cells and precursors by activating PML-dependent pathways. Similarly, interferons could act on APL cells, alone or in combination with all-trans retinoic acid (RA), especially if the PML/RAR alpha fusion transcript that results from the t(15; 17) is induced by interferon. We report here that PML is expressed at low levels or not expressed in normal circulating human monocytes, lymphocytes, and polymorphonucleate cells, but is markedly induced by interferon; that PML and PML/RAR alpha expression is augmented by interferon in the NB4 APL cell line, which carries the t(15; 17), and in APL blasts from patients; that interferon inhibits growth and survival of NB4 APL cells in cooperation with RA; that interferons alone have minimal maturation effect on NB4 cells; and, finally, that interferon gamma, but not alpha or beta, induces maturation and growth suppression of NB4 cells with de novo retinoid resistance, and partially restores RA response.

Cell Differentiation↗
PubMed1996

A family of Shc related proteins with conserved PTB, CH1 and SH2 regions.

Shc proteins are targets of activated tyrosine kinases and have been implicated in the transmission of activation signals to Ras. Upon phosphorylation, Shc proteins form stable complexes with cellular tyrosine-phosphorylated proteins and with the Grb2 adaptor protein. Two Shc isoforms of 52 and 46 kDa have been characterized. They share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal phospho-tyrosine binding domain (PTB). We report her ethe initial characterization of two Shc related human cDNAs: ShcB and ShcC. The ShcB and ShcC cDNAs code for proteins that are highly similar and share the same modular organization as Shc. PTB and SH2 domains of ShcB and ShcC have similar binding specificities in vitro and bind to activated EGFR in a phosphotyrosine-dependent manner. Based on these findings we propose to rename Shc as ShcA. Anti-ShcB and anti-ShcC antibodies recognize specific polypeptides of 52, 47 kDa (ShcB) and 54 kDa (ShcC) in mammalian cells. Since these two genes are predominantly expressed in specific brain tissues, these Shc family members may be involved in cell type-specific signaling, in the nervous system.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Quantification of PML-RAR alpha transcripts in acute promyelocytic leukaemia: explanation for the lack of sensitivity of RT-PCR for the detection of minimal residual disease and induction of the leukaemia-specific mRNA by alpha interferon.

The RT-PCR technique for the identification of the PML-RAR alpha fusion mRNA is widely used for the detection of minimal residual in acute promyelocytic leukaemia (APL). A positive result after remission induction is highly predictive of early relapse, but the vast majority of patients have no detectable disease by this technique after chemotherapy consolidation, despite the fact that many later relapse. We report a quantitative PCR technique for the PML-RAR alpha cDNA which was used to show that less than 1000 PML-RAR alpha molecules are obtained from 1 microgram of diagnostic bone marrow RNA derived from approximately 1 million APL blasts. The lack of sensitivity of currently employed RT-PCR methods may therefore be explained by their poor yield of PML-RAR alpha cDNA. Minor modifications to the reverse transcription procedure improved this yield 3 fold. Furthermore, expression of the leukaemia-specific transcript increased by approximately one order of magnitude after incubation of the patient's cells for 24 h in vitro with 100 iu/ml alpha interferon.

DNA, Neoplasm↗
PubMed1995

Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress.

Glucose 6-phosphate dehydrogenase (G6PD) is a housekeeping enzyme encoded in mammals by an X-linked gene. It has important functions in intermediary metabolism because it catalyzes the first step in the pentose phosphate pathway and provides reductive potential in the form of NADPH. In human populations, many mutant G6PD alleles (some present at polymorphic frequencies) cause a partial loss of G6PD activity and a variety of hemolytic anemias, which vary from mild to severe. All these mutants have some residual enzyme activity, and no large deletions in the G6PD gene have ever been found. To test which, if any, function of G6PD is essential, we have disrupted the G6PD gene in male mouse embryonic stem cells by targeted homologous recombination. We have isolated numerous clones, shown to be recombinant by Southern blot analysis, in which G6PD activity is undetectable. We have extensively characterized individual clones and found that they are extremely sensitive to H2O2 and to the sulfydryl group oxidizing agent, diamide. Their markedly impaired cloning efficiency is restored by reducing the oxygen tension. We conclude that G6PD activity is dispensable for pentose synthesis, but is essential to protect cells against even mild oxidative stress.

Animals↗
PubMed1995

The acute promyelocytic leukaemia-associated PML gene is induced by interferon.

The PML protein concentrates within discrete nuclear structures known as nuclear bodies, also called NDs or PODs, which contain several proteins including the interferon (IFN)-inducible SP100 product. The function of these structures remains elusive. We and others have shown recently that they represent specific targets for adenovirus and herpes simplex virus. This prompted us to investigate whether PML, like SP100, might be induced by IFN and to explore the role of PML in viral infection. Here we report that PML mRNA levels increase rapidly in response to interferon treatment. This accumulation of PML transcripts is a primary IFN response since it does not require de novo protein synthesis. The IFN-induced activation of the PML gene is accompanied by enhanced protein expression as revealed by immunolabelling. Both the intensity of the staining and the number of labelled structures increased upon interferon exposure. To probe the role of PML in IFN action, we compared the antiviral state established by alpha-interferon in embryonic fibroblasts (EFs) derived from null mutant mice for PML and from wild-type control mice. The resistance to viral infection conferred by IFN-alpha was identical in both PML+/+ and PMLm/m fibroblasts indicating that PML is not an essential mediator of the antiviral effect of interferon. We also noted that DNA-binding factors are normally activated by IFN in PMLm/m cells.

Base Sequence↗
PubMed1995

Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.

GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.

Abnormalities, Multiple↗
PubMed1995

Selection of a highly enriched population of retrovirus-infected human hematopoietic progenitor cells using SNL fibroblasts.

Retrovirus-mediated gene transfer into human hematopoietic progenitor cells for therapeutic or experimental purposes has proved difficult due to low and variable infection efficiency. To address this, we have developed an in vitro system for the selection and maintenance of a highly-enriched population of retrovirus-infected hematopoietic progenitor cells. Human umbilical cord CD34+ cells were cultured on SNL, a neo-containing murine fibroblast cell line used for embryonic stem cell culture. SNL-supported CD34+ cultures could be maintained with continuing blast cell and CFU-GM production for eight weeks, compared to four weeks in the absence of SNL. We then tested the ability of SNL to facilitate the selection in G418 of CD34+ cord cells infected with the neo-containing retrovirus, vsn-2. While all cells in the control cultures died within 14 days, vsn-2-infected CD34+ cells continued to proliferate, differentiate and produce CFU-GM for up to five weeks after infection. 100% of individually-plucked CFU-GM from such cultures were shown by PCR to be successfully infected. This approach should be useful for experimental work and, since it would diminish competitive repopulation between infected and uninfected progenitors, may also be utilized, with modification, for optimizing gene therapy protocols.

3T3 Cells↗
PubMed1995

Developmental analysis of murine Promyelocyte Leukemia Zinc Finger (PLZF) gene expression: implications for the neuromeric model of the forebrain organization.

Promyelocyte Leukemia Zinc Finger (PLZF) is a Kruppel-like zinc finger gene previously identified in a unique case of acute promyelocytic leukemia (APL) as the counterpart of a reciprocal chromosomal translocation involving the retinoic acid receptor alpha gene (RAR alpha). PLZF is highly conserved throughout evolution from yeast to mammals. To elucidate its role, we isolated the murine PLZF gene and studied its expression during embryogenesis. PLZF is expressed in an extremely dynamic pattern with transcripts appearing at E 7.5 in the anterior neuroepithelium and quickly spreading to the entire neuroectoderm until E 10. At E 8.5, PLZF is transcribed in most of the endoderm. During mid to late gestation PLZF is expressed in restricted domains of the developing CNS as well as in specific organs and body structures. We have focused our attention on the developing forebrain where PLZF is transcribed in a transverse, segment-like domain corresponding to the anterior pretectum, in the alarmost part of the dorsal thalamus, in the epithalamus, and in the hypothalamus along a defined longitudinal subdomain. Furthermore, PLZF is expressed in several segmentary boundaries, among them, the zona limitans intrathalamica. Combined analysis with other regionally restricted genes, such as Orthopedia and Dlx1, indicates that in the hypothalamus the PLZF domain is contained within that of Orthopedia and both are complementary to that of Dlx1. Our data suggest a role for PLZF in the establishment and maintenance of transverse identities, longitudinal subdomains, and interneuromeric boundaries, providing additional evidences in favor of the neuromeric organization of the forebrain.

Animals↗
PubMed1994

Monitoring of treatment outcome in acute promyelocytic leukemia by RT-PCR.

A rearrangement between the PML and RAR-alpha genes underlies the acute promyelocytic leukemia (APL)-specific t(15;17) translocation, leading to the production of a chimeric mRNA. Recent development of a reverse-transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha hybrid has proven useful for rapid diagnosis and monitoring of minimal residual disease (MRD) in APL patients. Preliminary studies in which the prognostic significance of RT-PCR was evaluated indicate that this test may identify patients at high risk of relapse.

Hematologic Diseases↗
PubMed1994

Monitoring of treatment outcome in acute promyelocytic leukemia by RT-PCR.

A rearrangement between the PML and RAR-alpha genes underlies the acute promyelocytic leukemia (APL)-specific t(15;17) translocation, leading to the production of a chimeric mRNA. Recent development of a reverse-transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha hybrid has proven useful for rapid diagnosis and monitoring of minimal residual disease (MRD) in APL patients. Preliminary studies in which the prognostic significance of RT-PCR was evaluated indicate that this test may identify patients at high risk of relapse.

Chromosomes, Human, Pair 15↗
PubMed1993

Absence of reverse transcription-polymerase chain reaction detectable residual disease in patients with acute promyelocytic leukemia in long-term remission.

Hybrid fusion genes are specific tumor markers of several leukemic subtypes. The use of reverse transcription-polymerase chain reaction (RT-PCR) to amplify chimeric cDNAs allows sensitive detection of the neoplastic clone for diagnostic and monitoring studies in these leukemias. Nonetheless, the clinical relevance of minimal residual disease (MRD) evaluation by PCR remains controversial. In this study, 9 patients (pts) with acute promyelocytic leukemia (APL) in long-term remission for 4 to 12 years were analyzed for the presence of MRD by RT-PCR amplification of the specific PML/RAR-alpha fusion gene. Seven pts had been treated with conventional chemotherapy (CHT) alone, 1 had undergone allogeneic bone marrow transplantation (BMT), and 1 autologous BMT as consolidation therapy after CHT. In 8 cases, the presence of the t(15;17) rearrangement could be documented in diagnostic BM specimens by cytogenetic and/or molecular analysis. A two-rounds "nested" RT-PCR assay with sensitivity levels of 1 in 10(5) was used to analyze BM samples collected at 32 to 141 months from the achievement of complete remission (CR). In no cases were residual PML/RAR-alpha transcripts detectable in these remission controls. All patients are in unmaintained CR at 48 to 154 months from CR and at 6 to 17 months from PCR evaluation. These results suggest that long-term survival of APL is associated with eradication of cells carrying the specific PML/RAR-alpha rearrangement, indicating that PCR negativity should be considered the therapeutic goal in these patients. Our findings further strengthen the clinical relevance of PCR monitoring studies in APL, as opposite to other leukemic subtypes (chronic myeloid leukemia and acute myeloid leukemia-M2) in which the prognostic significance of PCR evaluation is unclear.

Adult↗
PubMed1993

Human CD4 lymphocytes specifically recognize a peptide representing the fusion region of the hybrid protein pml/RAR alpha present in acute promyelocytic leukemia cells.

Fusion proteins present in leukemic cells frequently contain a new amino acid at the fusion point. We tested whether a peptide (BCR1/25) encompassing the fusion region of the hybrid molecule pml/RAR alpha, which is selectively expressed by acute promyelocytic leukemia (APL) cells, can be recognized by human T lymphocytes in vitro. CD4+ lymphocytes, at both polyclonal and clonal level, recognized peptide BCR1/25 in an HLA-DR--restricted fashion on presentation by autologous antigen-presenting cell (APC) or by APC expressing the HLA-DR11 restricting molecule. Control peptides corresponding to the normal pml and RAR alpha proteins were not recognized. One clone (DEG5) also exerted a high and specific cytotoxicity against autologous cells pulsed with BCR1/25. The autologous DE LCL containing a transduced pml/RAR alpha fusion gene and expressing a bcr1 type of the pml/RAR alpha hybrid protein induced the proliferation of DE anti-BCR1/25 T cell clones. It is concluded that the bcr1 type-pml/RAR alpha fusion protein of APL contains an antigenic site, absent from the normal parent molecules and recognized by human CD4+ lymphocytes.

Amino Acid Sequence↗
PubMed1992

Molecular evaluation of residual disease as a predictor of relapse in acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) is characterised by a unique fusion transcript, PML/RAR alpha. We tested for this transcript in 35 APL patients who were in apparent remission after various treatments. 11 of 13 patients who tested positive 4 months after achieving remission were in relapse 1-4 months later. All 22 patients who tested negative at 4 months were disease-free after a further 3 months to five years. The test may therefore prove useful in determining the need for additional treatment during clinical remission.

Carrier Proteins↗
PubMed1992

Molecular monitoring of the myl/retinoic acid receptor-alpha fusion gene in acute promyelocytic leukemia by polymerase chain reaction.

The acute promyelocytic leukemia (APL) t(15;17) translocation generates a myl/retinoic acid receptor-alpha (RAR-alpha) chimeric gene that is transcribed as a fusion myl/RAR-alpha messenger RNA. Using primer sets derived from RAR-alpha and myl cDNAs, we were able to amplify the breakpoint sites of the fusion transcripts of all 35 APL RNA samples by reverse polymerase chain reaction (PCR) and nested primer approach of two rounds of amplification. DNA fragments of different size were obtained according to the chromosome 15 breakpoints (intron 3-bcr 3; exon 6-bcr 2; and intron 6-bcr 1). bcr 1 and bcr 3 represent the regions of the myl locus most frequently involved among APL (48.5 and 34.2 of cases, respectively); bcr 3 constitutes 62.5% of cases among M3V as compared with 25.9% of M3 cases. The feasibility of monitoring the APL clone by PCR analysis in five APL patients who received different treatment (chemotherapy, all-trans-retinoic acid or bone marrow transplantation) was evaluated. In five of nine bone marrow samples of patients in complete remission, t(15;17)-positive cells could be detected by PCR analysis. We conclude that PCR amplification of the myl/RAR-alpha junctions represents the easiest and rapid method for diagnosis and monitoring of the APL clone.

Adolescent↗
PubMed1992

Characterization of the PML-RAR alpha chimeric product of the acute promyelocytic leukemia-specific t(15;17) translocation.

The acute promyelocytic leukemia 15;17 chromosomal translocation fuses the PML gene to the RAR alpha locus. The resulting chimeric gene encodes for a putative PML-RAR alpha fusion protein. PML is a putative transcriptional factor and RAR alpha is one of the nuclear retinoic acid receptors through which retinoic acid regulates gene expression. In this study, we investigated the retinoid binding and biochemical properties of the PML-RAR alpha protein by size exclusion high-performance liquid chromatography and immunoblot analysis and compared them with those of normal RAR alpha. The introduction of the expression vector PSG5/PML-RAR alpha into COS-1 cells led to high levels of expression of the PML-RAR alpha fusion protein. This protein was primarily localized in the nucleus and bound retinoids with the same affinity and specificity as the wild type RAR alpha receptor. The PML-RAR alpha fusion protein, but not the RAR alpha, was found in high molecular weight complexes with either itself or other nuclear factors. In the acute promyelocytic leukemia-derived cell line NB4, which contains the t(15;17) chromosomal marker, the PML-RAR alpha product was also found as a high molecular complex. The interaction of the PML-RAR alpha with itself or with other nuclear proteins may be important in understanding the role of the PML-RAR alpha fusion protein in promyelocytic leukemogenesis.

Carrier Proteins↗