Carlo Rumi

Quercetin potentiates the effect of adriamycin in a multidrug-resistant MCF-7 human breast-cancer cell line: P-glycoprotein as a possible target

This study demonstrates that the flavonoid quercetin (Q), a plant-derived compound with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of Adriamycin (ADR) on MCF-7 ADR-resistant human breast cancer cells. The effect of Q was dose-dependent at concentrations ranging between 1 and 10 μM. Since ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of Q and related flavonoids of Pgp activity in cytofluorographic efflux experiments with the fluorescent dye rhodamine 123 (Rh 123). Our results indicate that Q and 3-OMe Q (3′,4′,7-trimethoxyquercetin) but not the 3-rhamnosylglucoside of Q (rutin) inhibit the Pgp pump-efflux activity in a dose-related manner. Moreover, 10 μM Q reduces the expression of the immunoreactive Pgp in MCF-7 ADR-resistant cells as evaluated by cytofluorimetric assay. In conclusion, these findings provide a further biological basis for the potential therapeutic application of Q as an anticancer drug either alone or in combination with ADR in multidrug-resistant breast tumor cells.

Inhibitory effect of quercetin on OVCA 433 cells and presence of type II oestrogen binding sites in primary ovarian tumours and cultured cells

We investigated the effect of the flavonoid quercetin (Q) on the proliferation of the ovarian cancer cell line OVCA 433. Growth experiments demonstrated that Q exerted a reversible dose-dependent inhibition of cell proliferation in the range of concentrations between 10 nM and 10 microM. Two other flavonoids tested, rutin and hesperidin, were ineffective in inhibiting cell growth. Cell cycle analysis showed that the growth inhibitory effect of Q was due to a blocking effect in the GO/G1 phase. Using a whole cell assay with (6.7-3H) oestradiol (3H-E2) as tracer we demonstrated that OVCA 433 cells contain type II oestrogen binding sites (type II EBS). Competition analysis showed that Q competed for 3H-E2 binding to type II EBS while both rutin and hesperidin did not. Appreciable amounts of type II EBS were also detected in seven primary ovarian tumours. Our results suggest that Q may regulate ovarian cancer cell growth through a mechanism involving a binding interaction with type II EBS. This mechanism could also be active in vivo since primary ovarian tumours contain type II EBS.

Immune reconstitution after transplantation of autologous peripheral CD34+ cells: analysis of predictive factors and comparison with unselected progenitor transplants

The recovery of lymphocyte count, CD4+ and CD8+ T‐cell subsets, natural killer (NK) cells and CD19+ B‐cells was evaluated in a cohort of 15 patients receiving autologous CD34+ peripheral blood progenitor cells (PBPCs; group A) for haematological malignancies and in 20 patients transplanted with autologous unselected PBPCs (group B). Lymphocyte count recovered in both patient cohorts, being significantly lower in group A than in group B 1 (P = 0·008) and 2 months (P = 0·0035) after progenitor cell infusion. The repopulation of CD3+ T‐cells occurred more rapidly in group B than in group A (P = 0·034 on week 4); CD19+ B‐lymphocytes did not return to reference ranges in either group of patients. The count of CD4+ T‐lymphocytes remained < 200/μl during the study period in patients transplanted with CD34+ PBPCs, significantly lower than group B levels (P = 0·034 and P = 0·021 on weeks 4 and 8 respectively). CD8+ T‐cells increased rapidly in both groups; thus, the CD4 to CD8 ratio was severely reduced. CD4+ and CD8+ T‐cells displayed an activated phenotype in both groups of patients, co‐expressing the HLA‐DR antigen throughout the study period. NK cells followed a similar repopulation kinetics in both study groups, although their expansion was greater in group B than in group A (P = 0·014 on week 4). In the CD34+ group, post‐transplant administration of granulocyte colony‐stimulating factor predicted a faster lymphocyte recovery in multivariate analysis (P = 0·025); interestingly, the amount of passively transferred lymphocytes correlated inversely with time to achieve a lymphocyte count > 0·5 × 109/l (r = –0·63, P = 0·01). Further investigations are necessary to characterize T‐cell competence after transplantation of CD34+ PBPCs.

CD34-positive cells in human umbilical cord blood express nerve growth factor and its specific receptor TrkA

In this study, we investigated whether hematopoietic stem cells (HSC) and progenitors present in human cord blood can express nerve growth factor (NGF)-specific receptors, TrkA and p75. Our results showed a marked expression of TrkA and NGF in cord blood CD34(+) cells. A gradient of TrkA and NGF expression exists and is highest in cord blood CD34(+) cells, reduced in cord blood mononuclear cells (MNC) and minimal in mononuclear cells isolated from adult peripheral blood. Our findings suggest that NGF may play a role in the differentiation of hematopoietic progenitors and indicate a different requirement for NGF by immune cells, depending on their state of maturity.

CD34+/CD105+ cells are enriched in primitive circulating progenitors residing in the G0 phase of the cell cycle and contain all bone marrow and cord blood CD34+/CD38low/− precursors

A subset of circulating CD34+ cells was found to express CD105 antigen. Sorting experiments showed that most granulocyte–macrophage colony‐forming units (GM‐CFU) and burst‐forming units — erythroid (BFU‐E) were retained in the CD34+/CD105− fraction, whereas rare GM‐CFU/BFU‐E were generated from CD34+/CD105+ cells. Megakaryocytic aggregates were entirely retained in the CD34+/CD105+ fraction. Neutralizing doses of an anti‐TGF‐β1 antibody demonstrated CD34+/CD105+ cells capable of colony‐forming activity without any significant effect on CD34+/CD105− cells. Cloning of secondary colonies revealed that CD34+/CD105+ cells had a significantly higher secondary cloning efficiency than CD34+/CD105− cells. CD34+/CD105+ cells had a significantly higher long‐term culture‐initiating cell (LTC‐IC) frequency than CD34+/CD105− cells. Kinetic analysis showed that 75% of CD34+/CD105+ cells consisted of DNA 2n G0Ki‐67− cells whereas 82% of CD34+/CD105− were DNA 2n G1Ki‐67+ cells, and this latter subset showed a RNA content consistently higher than CD34+/CD105+ cells. CD34+/CD105+ progenitors were CD25+, whereas CD34+/CD105− contained a small CD25+ subset. Three‐colour analysis of bone marrow and cord blood CD34+ cells demonstrated that all the CD34+/CD38low/− primitive precursors were contained in CD34+/CD105+ cells. Extensive characterization of these CD105+ precursors indicated that they have biological properties associated with primitive haematopoietic precursors.

Hereditary thrombocytosis caused by MPLSer505Asn is associated with a high thrombotic risk, splenomegaly and progression to bone marrow fibrosis

Background The MPLSer505Asn mutation has been reported to be a cause of hereditary thrombocythemia. Recently, we detected this mutation in a large proportion of children with familial thrombocythemia, suggesting that in Italy the incidence of MPLSer505Asn mutation could be underestimated. Design and Methods We extended the search for this mutation to all patients with essential thrombocythemia who had a positive family history for thrombocytosis or essential thrombocythemia. We identified eight Italian families positive for the MPLSer505Asn mutation. Clinical and hematologic data were available for members of seven families, including 21 patients with a proven mutation and 20 relatives with thrombocytosis. Results Fifteen major thrombotic episodes, nine of which were fatal, were recorded among 41 patients. The thrombotic manifestation was stroke in four cases, myocardial infarction in seven cases, fetal loss in two cases, deep vein thrombosis of the leg in one case and Budd Chiari syndrome in one case. Almost all patients over 20 years old had splenomegaly and bone marrow fibrosis, while these were rarely observed in patients under 20 years old, suggesting that these manifestations are associated with aging. Finally, the life expectancy of family members with thrombocytosis was significantly shorter than that of members without thrombocytosis (P=0.003). Conclusions Patients with familial thrombocytosis caused by a MPLSer505Asn mutation have a high risk of thrombosis and, with aging, develop splenomegaly and bone marrow fibrosis, significantly affecting their life expectancy.

Differential sensitivity of leukemic and normal hematopoietic progenitors to the killing effect of hyperthermia and quercetin used in combination: role of heat-shock protein-70.

Autologous bone‐marrow transplantation (ABMT) is widely used in the treatment of acute leukemias where a matched sibling donor is not available for allogeneic transplantation. However, a major problem in ABMT is relapse, and ex vivo purging may be very important in preventing it. We show here that quercetin enhances the growth‐inhibitory effect of hyperthermia (HT) in AML (19 cases) and ALL (6 cases) leukemic blasts. Furthermore, the inhibitory effect of this combined treatment resulted in leukemic‐cell apoptosis. On the contrary, normal hematopoietic progenitors were neither growth‐inhibited nor induced to apoptosis by HT‐plus‐quercetin treatment. To explain this difference in sensitivity of leukemic and normal hematopoietic progenitors, we analyzed the effect of quercetin on heat‐induced expression of heat‐shock protein‐70 (HSP‐70), which has been shown to be important in regulating thermosensitivity. We found that quercetin inhibits heat‐induced HSP‐70 expression both at protein and at mRNA levels in AML and ALL blasts. In normal CD34+ progenitors, the combined treatment with HT and quercetin did not reduce HSP‐70 expression and did not induce cell apoptosis. Considering the difference in heat sensitivity of normal CD34+ and leukemic progenitors in the presence of quercetin, the combined use of HT and quercetin could constitute a purging protocol for ABMT. Int. J. Cancer 73:75–83, 1997.

Further investigations on the expression of HLA-DR, CD33 and CD13 surface antigens in purified bone marrow and peripheral blood CD34± haematopoietic progenitor cells

Summary. We evaluated the HLA‐DR, CD33 and CD13 antigen expression on CD34± haematopoietic progenitor cells (HPC) isolated from the bone marrow (BM) and peripheral blood (PB) of normal donors. The majority of both BM and PB CD34± HPC expressed CD13 and HLA‐DR. The coexpression of CD34 and CD33 was found in a minor CD34± subset. After 7 d of culture in the presence of interleukin‐3 and granulocyte‐macrophage colony‐stimulating factor, CD33 expression was detected in about 50% of HPC. At this point CD34 antigen expression was lost and CD13 and HLA‐DR expression was partially lost. After 14 d of culture, the majority of HPC were CD33±. HPC maintained the capacity to generate colony forming unit granulocyte‐macrophage but they lost the capacity to generate burst forming unit‐erythroid. A correlation was found between the percentage of CD34±/HLA‐DR± cells and the total number of colony forming cells in unfractionated samples from BM and PB of patients with malignancies. These studies demonstrate that, in normal conditions, only a minor subset of CD34± cells coexpress CD33 antigen either in BM or in PB and CD33 antigen is a lineage marker which is coexpressed with HLA‐DR and CD13 on a progenitor committed to the granulocytic‐macrophagic lineage.

Induction of CD69 antigen on normal CD4+ and CD8+ lymphocyte subsets and its relationship with the phenotype of responding T‐cells

We evaluated phenotype and apoptotic status of normal CD4+CD69+ and CD8+CD69+ peripheral blood T-lymphocytes after short-term challenge with escalating concentrations of phytohemagglutinin (PHA). The frequency of CD69-coexpressing CD4+ and CD8+ T-cells and CD69 staining intensity increased following T-cell mitogenic stimulation; these changes were proportional to PHA concentration in culture medium. A considerable fraction of lymphocytes underwent blast transformation, displaying increased forward and side scatter signals. Interestingly enough, PHA-responsive T-cells exhibited a predominantly CD25negCD38negTCRalphabetapos phenotype; APO-1/Fas antigen (CD95) could be detected on a minority of activated CD69+ T-cells. A considerable proportion of CD69+ lymphocytes expressed intracellular perforin; in addition, an average 16+/-6% CD69+ T-lymphocytes were apoptotic after 4 h of stimulation, as evaluated by 7-amino-actinomycin-D staining and by annexin-V binding. CD69+ activated lymphocytes comprise phenotypically heterogeneous cell subpopulations potentially devoted to diverse immunological functions, i.e., proliferation, apoptosis, or cell cytotoxicity; moreover, our findings indicate that CD69 expression is proportional to the intensity of the activating stimulus and that the capacity to upregulate CD69 antigen following short-term mitogenic challenge may be restricted to unactivated CD38negCD25negTCRalphabetapos T-lymphocytes.

Vernal Keratoconjunctivitis: A Model of 5q Cytokine Gene Cluster Disease

Clinical studies of vernal keratoconjunctivitis (VKC) patients show that total IgE serum levels are increased even in the absence of IgE antibodies to common allergens. Activated eosinophils are also a constant feature of VKC at both the circulation (cytofluorimetry) and tissue (tear cytology and conjunctival scrapings) levels. Moreover, allergen challenge induces a prolonged inflammatory reaction with a prevalent participation of eosinophils, lymphocytes and possibly basophils. Immunohistochemical studies of VKC biopsies show a multicellular inflammatory infiltrate with prevalence of activated eosinophils, mast cells and CD4 lymphocytes in both epithelium and subepithelium. Mediator studies indicate that eosinophil products (eosinophil peroxidase, eosinophinal cationic protein and eosinophil-derived neurotoxin/eosinophil protein X) are increased in both serum and tears, where tryptase and interleukin (IL)-5 are also detectable in higher amounts than in controls. On the basis of these findings, we postulate that VKC can represent a phenotypic model of up-regulation of the cytokine gene cluster on chromosome 5q which through its products (IL-3, IL-4, IL-5 and granulocyte/macrophage-colony-stimulating factor) regulates Th2 prevalence, IgE production as well as mast cell and eosinophil growth and function in VKC.