Molecular mechanism of hematopoietic stem cell binding to the supportive stroma.
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Animals↗
Biomedical subjects
M Tavassoli
Publications and source records attributed to M Tavassoli.
Murine spleen culture: homing of hemopoietic progenitor cells to spleen is not mediated by a similar mechanism to that in marrow.
We have previously shown that in long-term bone marrow cultures (LTMC) the specific recognition and binding of hemopoietic stem cells to stroma, which we call "homing," is mediated by a recognition mechanism involving a surface membrane lectin with galactosyl and mannosyl specificities. Subsequent in vivo studies in lethally irradiated mice confirmed that homing to the marrow similarly involves a galactosyl- and mannosyl-specific recognition mechanism. However, these in vivo studies suggested that homing of hemopoietic progenitor cells to spleen was based upon a different molecular recognition mechanism. In the present study splenic homing was investigated in a cell culture system composed of an adherent layer of splenic stromal cells inoculated with stroma-free stem cells from the supernate of LTMC. In this system, splenic stroma supported proliferation and differentiation of hemopoietic precursors for a few weeks. When stem cells were added to the cultures in the presence or absence of inhibitory concentrations of neoglycoprotein reagents specific for galactosyl, mannosyl, or fucosyl lectins, the pattern of production of total cells, pluripotential stem cells (CFU-S), and granulocyte-macrophage committed progenitors (CFU-GM) remained the same. These data support our in vivo observations that homing of stem cells to splenic stroma is not mediated by a surface lectin with galactosyl and mannosyl specificities as it is in bone marrow, but rather by a different molecular mechanism.
Animals↗
Interaction of late murine erythroid progenitors and stroma involves a recognition mechanism with fucosyl specificity.
We have previously reported that the specific recognition and binding of murine hemopoietic progenitors spleen colony-forming units (CFU-S) and granulocyte-macrophage CFU (CFU-GM) to hemopoietic stroma is dependent upon a membrane recognition system with galactose and mannose specificities. By using synthetic neoglycoproteins with galactose, mannose, or fucose covalently bound to bovine serum albumin (BSA) in standard long-term bone marrow cultures (LTBMC), galactosyl-BSA (gal-BSA) and mannosyl-BSA (man-BSA) but not fucosyl-BSA (fuc-BSA) inhibited the binding of CFU-S and CFU-GM to the stromal layer. In the present work it was shown that binding of erythroid burst-forming units (BFU-E) to stroma in standard LTBMC is also inhibited by gal-BSA and man-BSA. We then studied a different system of LTBMC that favored erythropoiesis and allowed the production of erythroid CFU (CFU-E) for 4 weeks. In the presence of the fuc-BSA as well as gal-BSA and man-BSA, total cell production and CFU-E production were halted in the supernate as well as the adherent layer. These results indicate the presence of a fucosyl recognition system on the surface of the late erythroid precursors, CFU-E.
Animals↗
Purification and partial characterization of membrane-homing receptors in two cloned murine hemopoietic progenitor cell lines.
Selective seeding of bone marrow by intravenously transplanted hemopoietic cells depends on the homing receptors of these cells. The receptors are membrane lectins with galactosyl and mannosyl specificities. To purify these lectins, cell membrane was fractionated from two cloned multipotential (B6STU) and bipotential (FDCP-1) hemopoietic progenitor cells. The membrane was solubilized and its proteins were labeled with 125I. The proteins were subjected to affinity column chromatography using galactosyl and mannosyl groups linked to agarose beads. Elution with D-galactose or D-mannose led to specific elution of a single sharp radioactive peak which constituted a constant fraction of membrane proteins. This peak was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by disuccinimidyl suberate cross-linking technique and appeared to have a Mr of 110,000. Under reducing conditions, it consisted of two components with a Mr of 87,000 and 23,000. Treatment with endoglycosidase F indicated about 5% carbohydrate content. Purification of these homing receptors has opened an avenue for the development of immunologic and molecular biologic probes that may help further elucidate the mechanism of homing regulation.
Animals↗
Desialylation of transferrin by liver endothelium is selective for its triantennary chain.
Liver endothelium can remove and transport the glycoprotein transferrin (TF). During this process the molecules are desialylated; however, in contrast with other such glycoproteins, for example caeruloplasmin, only half of transported TF is desialylated. To explore which component of TF is desialylated, we double-labelled fully sialylated TF with [3H]sialic acid residues and a 125I-protein moiety. This was then 'chased' through purified liver endothelium in pulse-chase experiments. Endothelium-conditioned TF was fractionated on an RCA120 affinity column into sialylated and desialylated components. Each component was then re-fractionated on a concanavalin A affinity column, which separates the glycoprotein according to the branching pattern of its glycan chain. The desialylated fraction was eluted only as a triantennary component, whereas the non-desialylated fraction consisted only of bi- and tetra-antennary chains. The significance of this selective desialylation of triantennary chain of TF in the subsequent metabolism of its iron content and its possible role in the pathogenesis of alcohol-induced hepatic siderosis are discussed.
Animals↗
The role of endosomal traffic in the transendothelial transport of ceruloplasmin in the liver.
Through a process resembling receptor-mediated endocytosis, liver endothelium binds and internalizes the plasma glycoprotein ceruloplasmin (CP) on the luminal side. The protein is then transported via a vesicular system to the albuminal side where it is externalized to the space of Disse. In its path, the glycoprotein is fully desialylated. To determine if the endosomal compartment is involved in this transport, we used endosomal inhibitors NH4Cl, ethylamine as well as monensin to quantitatively measure the magnitude of radiolabeled CP transport across purified liver endothelial cells. All three reagents inhibited the transport of CP and its discharge by endothelium. The magnitude of inhibition was dose-related for all three reagents. We conclude that the endosomal compartment is involved in the transendothelial transport of CP across the liver endothelium.
Ammonium Chloride↗
Proteoglycan synthesis by hematopoietic progenitor cells.
The synthesis of proteoglycans (PG) by hematopoietic stromal cells has been reported. But PG synthesis by hematopoietic progenitor cells has not been explored. We have studied synthesis, cellular distribution, and molecular characteristics of PG by a cloned interleukin-3 (IL-3)-dependent hematopoietic progenitor cell line, FDCP-1, which is cloned from murine long-term marrow cultures. Under appropriate conditions the cell can differentiate into granulocytes and macrophages, and therefore, can be considered CFU-GM equivalent. The pattern of PG synthesis was studied by 35SO4 labeling. FDCP-1 cells actively synthesize PG, which are distributed in the intracellular, membrane-associated (MP), and extracellular pools. After purification of the 35S-labeled material by ion-exchange and gel filtration techniques, a single chondroitin sulfate-PG (CIS-PG) was observed to be present in the three studied pools. By Sepharose CL-4B chromatography, this PG has a Kav of 0.47, which after alkaline treatment is shifted to a Kav of 0.67. This indicates the proteoglycan nature of the 35SO4-labeled material. The MP CIS-PG is not stable. It is released to the culture medium where it is subsequently processed. However, in the presence of hematopoietic stromal cells D2X, the stability of MP proteoglycan of FDCP-1 cells is enhanced, suggesting that the synthesis of PG by progenitor cells and its accumulation in the membrane may have a role in the interaction between progenitor and stromal cells.
Animals↗
Comparison of desialylation of rat transferrin by cellular and non-cellular methods.
We have previously shown that the liver endothelium can desialylate the glycoprotein transferrin (Tf). In the present work we provide evidence that asialotransferrin obtained by this means behaves differently on Ricinus communis agglutinin (RCA120) lectin affinity chromatography from asialotransferrin obtained by either neuraminidase treatment or acid hydrolysis. Purified rat transferrin was radiolabelled either with 125I (protein moiety) or with 3H (sialyl residues), and subsequently saturated with iron. It was then passed through an RCA120-agarose column to isolate the fully sialylated component. Sialylated Tf was then desialylated either by incubation with purified rat liver endothelium or, in vitro, by neuraminidase treatment or by acid hydrolysis. The protein was again subjected to RCA120 column chromatography. Although both neuraminidase treatment and acid hydrolysis almost completely desialylated the glycoprotein (as evidenced by near absence of 3H label), the glycoprotein was not retained by the RCA120-agarose column. By contrast, liver endothelium partially desialylated the glycoprotein, but this desialylated fraction was retained by the RCA120-agarose column. These results suggest that desialylation with neuraminidase or acid hydrolysis may be inadequate for functional studies of asialotransferrin.
Animals↗
Erythropoiesis in murine long term marrow cultures.
We utilized a system of long-term bone marrow cultures that favored erythropoiesis and allowed the production of CFU-E for four weeks. Certain conditions of culture were crucial for erythropoiesis to occur. These conditions have been discussed. This system should allow further in vitro study of the regulation of erythropoiesis.
Animals↗
c-erbB-2/c-erbA co-amplification indicative of lymph node metastasis, and c-myc amplification of high tumour grade, in human breast carcinoma.
A panel of 73 samples, including 52 primary breast carcinomas, 10 normal breast tissues and 11 axillary lymph nodes, has been analysed for the presence of amplifications and gross structural alterations, in the oncogenes c-erbB-2, c-erbA, c-myc, N-myc, c-mos and c-Ha-ras. The tumours were also classified, graded and staged histopathologically and their DNA ploidy (42 samples) was determined by flow cytometry. Three breast cancer cell lines (MCF7, ZR-75-1 and T47D) were also included in the study. Amplification of c-erbB-2 was detected in 28% of the tumours, of which 91% had an increased steady-state level of c-erbB-2 mRNA. Amplification of c-erbA was found in 23% of tumours and was always associated with the amplification of c-erbB-2. Ten out of 12 (83%) tumours which had c-erbB-2 and c-erbA co-amplification had metastasised to axillary lymph nodes (P less than 0.006). However, the human thymidine kinase gene, which is present at the same chromosomal location as these two oncogenes (17q21-22), was amplified in only tw tumours. Amplification of c-myc was detected in 21% of the tumours studied, of which 82% (P less than 0.005) were of histopathological grade 3 and none were of grade 1. Flow cytometry showed that 90% (P less than 0.01) of the analysed tumours with c-erbB-2 and c-erbA co-amplification, and 70% (P less than 0.1) of those with c-myc amplification were DNA aneuploid. This study demonstrates the potential value of c-myc amplification in the assessment of the tumour grade, rather than metastatic potential; and of the co-amplification of c-erbB-2 and c-erbA as a strong indicator of metastatic potential, rather than tumour grade.
Breast Neoplasms↗
Endothelial mediation is necessary for subsequent hepatocyte uptake of transferrin.
The authors previously have reported on the presence of transferrin (TF) receptors on liver endothelial cells and have shown that hepatic uptake of transferrin-iron (TF-Fe) complexes in the liver is mediated by the endothelium. We now provide evidence that this endothelial cell mediation may be necessary for hepatocyte uptake of TF-Fe complexes. Transport of TF-Fe from endothelial cell to hepatocyte was studied in mixed cell suspensions in which radiolabeled TF-Fe complexes were incubated at 37 degrees C with the two cell populations purified and then mixed in equal ratios. The mixtures were then refractionated at various times after incubation and cell-associated radioactivities measured. Radiolabeled TF was rapidly taken up by the endothelial cell fraction, but radioactivity began to decline in this fraction as it increased in the hepatocyte fraction. In double labeling experiments with 125I-TF-59Fe, both radiolabels moved across the endothelium in parallel fashion, indicating that Fe remains associated with TF during transcytosis. However, in hepatocytes the two radiolabels became dissociated, with Fe remaining cell-associated and TF being recycled. Hepatocyte uptake of processed TF was partially inhibitable by galactan and asialofetuin, indicating that hepatocyte uptake may occur via asialoglycoprotein receptors of hepatocyte.
Animals↗
Use of phenytoin in healing of war and non-war wounds. A pilot study of 25 cases.
Nineteen patients with war-related missile wounds and six with refractory civilian ulcers were treated with topical phenytoin sodium powder daily for up to 4 weeks. The mean healing time was 2 weeks for missile wounds, compared to historical controls requiring 6-8 weeks. Healing time was 4 weeks for civilian ulcers that had been unresponsive to any treatment over the previous 5 months. Twenty-two patients had complete healing, three required skin grafts. Wider use of this safe, inexpensive, readily available and easy-to-use agent is suggested because of its positive effect on wound healing and rapid pain relief.
Clinical Trials as Topic↗
Characterization of membrane homing receptors in two cloned murine hemopoietic progenitor cell lines.
To characterize homing receptors that are responsible for the recognition and specific binding of hemopoietic progenitor cells to the stroma, we synthesized and 125I-labeled a number of neoglycoproteins. We used these neoglycoproteins as ligand to detect receptors on the membrane of two cloned murine hemopoietic progenitor cell lines, B6SUT and FDCP-1. Both cell lines demonstrated membrane receptors with galactosyl and mannosyl, but not fucosyl, specificities. B6SUT galactosyl receptors showed a single receptor population with a Kd of about 2.3 X 10(-7) M and 10(6) receptors per cell. Mannosyl receptors demonstrated two components with high and low affinities respectively with Kd of about 2.5 X 10(-8) M and 1.0 X 10(-7) M, and respectively about 7.4 X 10(5) and 3.7 X 10(5) receptors per cell. Comparable data were also obtained for FDCP-1. Displacement experiments indicated that radioactive ligands bound to receptors could be increasingly displaced by homologous cold ligand giving typical sigmoid-shaped curves. Cold mannosyl probe could also displace radioactive galactosyl probe in a similar manner, but cold galactosyl probe displaced radioactive mannosyl ligand with a curve demonstrating two phases, further suggesting two receptor components for the mannosyl ligand. Mature murine neutrophils and red cells as well as human neutrophils, monocytes, and red cells showed no receptors. The functional significance of these receptors in binding to stromal cells was demonstrated by quantitation of the binding of 51Cr-labeled progenitor cells to the cloned stromal cell line, D2X, before and after enzymatic removal of various carbohydrate residues of membrane glycoconjugates. Enzymatic removal of galactosyl and mannosyl, but not fucosyl, residues almost totally eliminated the binding. The findings strongly suggest that these homing receptors are present on the surface of early hemopoietic progenitor cells. With maturation the cells lose their receptors, so that mature cells can be released into the blood stream.
Animals↗
A minibead method for detection of membrane lectins.
Membrane lectins are being increasingly implicated in many biological phenomena. Previous methods for detection of these substances are applicable only to homogeneous cell populations. We have now developed a method that permits morphological identification of lectin-bearing cells in heterogeneous cell populations. Amide-modified latex minibeads (0.345 or 0.532 micron) were activated with glutaraldehyde and then covalently bound to p-aminophenyl derivatives of various sugars. When the probe thus constructed was incubated with cell systems known to bear well-defined membrane lectins (galactosyl receptors in hepatocytes, mannosyl receptors in macrophages), binding occurred and could be visualized by scanning electron microscopy. Binding was inhibited in the presence of excess soluble sugar, indicating the specificity of reaction. Incubation of a mixture of two different-sized probes with two different cell types led to segregation of the probes. This method also permits semiquantification of binding.
Animals↗
Electron microscopic identification of hemopoietic progenitor cells by exploiting their sugar-recognizing receptors using a newly developed minibead technique.
To identify hemopoietic progenitor cells in electron microscopic preparations we exploited the presence of "homing receptors" with galactosyl, mannosyl, and fucosyl specificities on their membrane surfaces. Amide-modified latex minibeads were covalently linked to the para-aminophenyl derivative of galactose, mannose, and fucose in pyranose form. Incubation of these probes with murine bone marrow cells led to specific binding of probes to a small proportion of marrow cells, comprising 0.5%, 0.6%, and 0.4% of the mononuclear marrow cell population, respectively, for galactosyl, mannosyl, and fucosyl probes. Specificity of the binding was demonstrated by inhibition in the presence of excess soluble sugars. The cells binding the probes were small, 4-5 microns, with characteristics similar to those described for progenitor cells. The implications of this technique for purification of hemopoietic progenitor cells are discussed.
Animals↗
Purification and characterization of ceruloplasmin receptors.
Membrane receptors for ceruloplasmin were isolated by affinity chromatography using CP bound to CNBr-activated Sepharose 4B. The receptor was obtained in the form of a single sharp peak which when collected, concentrated and run on SDS-PAGE provided a single band with a molecular weight of 35,000. Treatment with endoglycosidase F reduced this molecular weight by 3%. It is concluded that ceruloplasmin receptors in this cell system is a membrane protein containing 3% carbohydrate with a total molecular weight of 35,000. It is possible that this is a monomer of a higher molecular weight protein.
Animals↗
DMSO induced modulation of c-myc steady-state RNA levels in a variety of different cell lines.
The nuclear proto-oncogene c-myc is believed to play a regulatory role in eukaryotic cellular growth and differentiation. Several studies have demonstrated rapid down-regulation of the steady-state levels of c-myc RNA during DMSO induced differentiation of the human 'promyelocytic' cell line, HL-60. However, little is known about the effect of DMSO on c-myc regulation in cells which are not induced to differentiate by DMSO. We have examined the effect of DMSO on c-myc RNA levels in a number of cell lines with different lesions in the c-myc gene, including those which do not differentiate in response to DMSO treatment. Here we demonstrate that DMSO induces a rapid, but transient, reduction in the steady-state level of c-myc RNA in human 'erythroid' K562 cells, Burkitt lymphoma lines Daudi and Raji, human T cell lymphoblastoid line CEM and mouse lymphoma line L1210. However, DMSO treatment does not produce a similar effect in the human colon adenocarcinoma cell line COLO 320 or HeLa epithelial cervical carcinoma cells. These observations demonstrate that the DMSO induced modulation of c-myc RNA levels is a more common phenomenon than previously recognised, and is not necessarily correlated with either the induction of cellular differentiation or growth arrest.
Cell Differentiation↗
Localization of megakaryocytes in the bone marrow.
In the bone marrow, megakaryocytes are located in the extravascular space, applied to the abluminal surface of endothelium. In this position, they send cytoplasmic projections into the lumen. Some of these projections are organelle free and may serve to anchor the cell to the endothelium. They could also serve to monitor the circulation and to receive information as to the requirement of the body for platelet formation. Megakaryocytes also send organelle containing projections into the lumen. This may be an early step in the migration of these cells into the lumen or, alternatively, part of the proplatelet formation. These proplatelets are 2.5 x 120 microns elongated structures that penetrate the lumen and can each subsequently make 1000 platelets. Each megakaryocyte can make six to eight proplatelets. In the perisinal position, megakaryocytes may subserve an adventitial function as well; many blood cells can then take a transmegakaryocytic route to reach the endothelium and enter the circulation.
Animals↗