Ligation of the CD44 adhesion molecule reverses blockage of differentiation in human acute myeloid leukemia.

Blockage in myeloid differentiation characterizes acute myeloid leukemia (AML); the stage of the blockage defines distinct AML subtypes (AML1/2 to AML5). Differentiation therapy in AML has recently raised interest because the survival of AML3 patients has been greatly improved using the differentiating agent retinoic acid. However, this molecule is ineffective in other AML subtypes. The CD44 surface antigen, on leukemic blasts from most AML patients, is involved in myeloid differentiation. Here, we report that ligation of CD44 with specific anti-CD44 monoclonal antibodies or with hyaluronan, its natural ligand, can reverse myeloid differentiation blockage in AML1/2 to AML5 subtypes. The differentiation of AML blasts was evidenced by the ability to produce oxidative bursts, the expression of lineage antigens and cytological modifications, all specific to normal differentiated myeloid cells. These results indicate new possibilities for the development of CD44-targeted differentiation therapy in the AML1/2 to AML5 subtypes.

CD44 targeting reduces tumour growth and prevents post-chemotherapy relapse of human breast cancers xenografts.

CD44 is a marker of tumour-initiating cells and is upregulated in invasive breast carcinoma; however, its role in the cancer progression is unknown. Here, we show that antibody-mediated CD44-targeting in human breast cancer xenografts (HBCx) significantly reduces tumour growth and that this effect is associated to induction of growth-inhibiting factors. Moreover, treatment with this antibody prevents tumour relapse after chemotherapy-induced remission in a basal-like HBCx.

CD44 ligation induces apoptosis via caspase- and serine protease-dependent pathways in acute promyelocytic leukemia cells.

We have recently reported that ligation of the CD44 cell surface antigen with A3D8 monoclonal antibody (mAb) triggers incomplete differentiation and apoptosis of the acute promyelocytic leukemia (APL)-derived NB4 cells. The present study characterizes the mechanisms underlying the apoptotic effect of A3D8 in NB4 cells. We show that A3D8 induces activation of both initiator caspase-8 and -9 and effector caspase-3 and -7 but only inhibition of caspase-3/7 and caspase-8 reduces A3D8-induced apoptosis. Moreover, A3D8 induces mitochondrial alterations (decrease in mitochondrial membrane potential DeltaPsi m and cytochrome c release), which are reduced by caspase-8 inhibitor, suggesting that caspase-8 is primarily involved in A3D8-induced apoptosis of NB4 cells. However, the apoptotic process is independent of TNF-family death receptor signalling. Interestingly, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) decreases A3D8-induced apoptosis and when combined with general caspase inhibitor displays an additive effect resulting in complete prevention of apoptosis. These results suggest that both caspase-dependent and serine protease-dependent pathways contribute to A3D8-induced apoptosis. Finally, A3D8 induces apoptosis in all-trans-retinoic acid-resistant NB4-derived cells and in APL primary blasts, characterizing the A3D8 anti-CD44 mAb as a novel class of apoptosis-inducing agent in APL.

Induction in rats of paranasal sinus carcinomas with radioactive cerium chloride.

A colloidal suspension of radioactive cerium stabilized in the form of hydroxide was inoculated into young Sprague-Dawley rats near the maxillary sinus. Well-differentiated epidermoid carcinomas of the paranasal sinus were obtained in 8 of 12 rats and in 8 of 9 animals that liver more then 200 days. Therefore, this istope was highly effectivein inducing tumors at the site of injection. The uniformity of the histologic type of tumors induced under these experimental conditions was remarkable.

Ectopic hematopoiesis in peritoneal tumor nodules induced by the myeloproliferative sarcoma virus in DBA/2 mice.

Tumor nodules composed of fibroblasts, large undifferentiated cells, granulocytes, and small lymphocytes develop in the spleens of adult DBA/2 mice infected with the myeloproliferative sarcoma virus (MPSV). They spread thereafter in the organism, and at the terminal stage of the disease they are especially numerous on the peritoneal membrane. The present study, performed on those tumor nodules to avoid contamination by exogenous hematopoietic cells, demonstrated that they were sites of granulopoiesis, which may have occurred via the local differentiation of granulomacrophage precursor cells (GM-CFC) and perhaps also from pluripotent hematopoietic stem cells, since these two populations were present in the tumor nodules (25 +/- 11 and 13 +/- 10, respectively, per 5-10(5) cells). Almost all (88%) those GM-CFC were able to clone in vitro without added colony-stimulating factor. A comparative study with the Moloney murine sarcoma virus-induced tumor indicated that the local production of hematopoiesis-stimulating factors was not sufficient to allow such ectopic granulopoiesis. These results imply the presence of a specific hematopoietic microenvironment in the MPSV-induced tumor nodules.

Increased synthesis of extracellular spleen glycosaminoglycans in an experimental myeloproliferative syndrome.

The changes occurring in the hematopoietic extracellular matrix in an experimental myeloproliferative syndrome were explored by comparing the glycosaminoglycan (GAG) composition of normal mouse spleens and spleens infected with myeloproliferative sarcoma virus (MPSV). Large quantities of hyaluronate and of sulfated GAGs accumulated in the extracellular matrix of infected spleens, as shown by histoimmunoassay and alcian blue staining, respectively. The splenic GAGs were either labeled with 35S-sulfate injected in vivo or unlabeled. The spleens were fractionated to separate hematopoietic cells from the stromal component containing extracellular matrix material and fibroblasts, and the GAGs were extracted from each fraction. Specific degradative treatments and electrophoresis indicated that sulfated GAGs were mostly chondroitin sulfate and heparan sulfate. Three hours after in vivo injection of 35S-sulfate, the amount of 35S-GAGs was increased approximately fivefold per mg stromal proteins. The bulk of these 35S-GAGs (70%) was recovered in the stromal fraction. The higher amount of sulfated GAGs in leukemic spleen was due both to the presence of more producer cells (infected fibroblasts and hematopoietic cells) and to a stimulation of GAG synthesis per cell, as evidenced 35S-labeling in in vitro experiments. Chondroitin sulfate was the main sulfated GAG present in the culture medium of both hematopoietic and fibroblastic cells and in the pericellular material released by trypsin from fibroblastic cells. High amounts of chondroitin sulfate, which has a possible role in the detachment of hematopoietic cells from the stromal cells, may favour the release of hematopoietic cells from the spleen into the peripheral blood. Heparan sulfate was produced by fibroblastic cells and it was principally present in their pericellular material. Considering the capacity of heparan sulfate to retain cytokines, as demonstrated by others in vitro, large amounts of heparan sulfate may result in the retention of large amounts of the cytokines, which production is enhanced in the infected spleen. This phenomenon may contribute to promote the hematopoietic stem cell proliferation characteristic of the MPSV-induced myeloproliferative disease.

A study of added GM-CSF independent granulocyte and macrophage precursors in mouse spleen infected with myeloproliferative sarcoma virus (MPSV).

A subpopulation of granulocyte and macrophage precursors (GM-CFUc) differentiating in the agar colony technique of Bradley and Metcalf into mature granulocytes and macrophages, without the addition of granulocyte and macrophage colony stimulating factor (GM-CSF), can be detected in MPSV infected mice. These precursors were detected 5 days after virus infection, reaching a maximal concentration of 1/10,000 spleen or bone marrow cells, 25 days after viral infection. The number of the added GM-CSF independent GM-CFUc was linearly correlated with the number of seeded MPSV hematopoietic cells. No GM-CSF producing cells could be detected in the MPSV spleen using normal bone marrow GM-CFUc as responder cells. Study of the GM-CSF sensitivity of the GM precursors has demonstrated the existence of two GM-CFUc populations in the MPSV spleen: a) a GM-CSF dependent population with a GM-CSF sensitivity similar to that of normal GM-CFUc b) a GM-CFUc population which differentiated in the absence of detectable amount of GM-CSF and of which differentiation was not affected by the addition of progressive amounts of GM-CSF. A possible model explaining these results is proposed.

Effects of anti-CD44 monoclonal antibody on adhesion of erythroid leukemic cells (ELM-I-1) to hematopoietic supportive cells (MS-5): CD44, but not hyaluronate-mediated, cell-cell adhesion.

Cocultivation of erythroid leukemic cells (ELM-I-1) with hemopoietic supportive cells (MS-5) resulted in a specific adhesion of ELM-I-1 cells to MS-5 cells. This phenomenon was designated as rosette formation. After induction of differentiation of ELM-I-1 cells, rosette formation was reduced, and no rosette formation was observed between erythrocytes and MS-5 cells. Studies on anti-adhesion molecule antibody treatment have revealed that CD44 plays a key role in rosette formation. Expression of CD44 on (the membrane of) ELM-I-1 cells was reduced after differentiation, and no CD44 expression was detected on erythrocytes. CD44 was also expressed on MS-5. Hyaluronate is known as the ligand of CD44, but neither hyaluronidase treatment nor addition of excess hyaluronate to the assay system affected rosette formation. These data indicate that hyaluronate is not responsible for rosette formation. Anti-CD44 antibody (KM81), which recognized the hyaluronate binding site of CD44, inhibited rosette formation. But other monoclonal antibodies against different epitopes except for the hyaluronate binding site, even those against CD44's hyaluronate binding site, did not inhibit rosette formation. Thus, rosette formation between MS-5 cells and ELM-I-1 cells is mediated by CD44 but not by the hyaluronate binding site of CD44.

In vitro chronopharmacology of recombinant mouse IL-3, mouse GM-CSF, and human G-CSF on murine myeloid progenitor cells.

Circadian changes in in vitro pharmacodynamic effects of recombinant mouse interleukin-3 (rmIL-3), rm granulocyte-macrophage colony-stimulating factor (rmGM-CSF), and recombinant human G-CSF (rhG-CSF) were investigated in 418 male B6D2F1 mice. Seven distinct experiments were staggered from July to December 1991. All mice were standardized for 3 weeks with a lighting schedule consisting of 12 hours of light and 12 hours of dark (LD12:12). In each experiment, bone marrow was sampled from separate groups of nine to 10 mice each every 4 hours for 24 hours. Data were analyzed with analysis of variance (ANOVA) and Cosinor. This latter method computes the probability of rhythm detection and its parameters. Femoral myeloid progenitors were quantified using the colony-forming units granulocyte/macrophage (CFU-GM) assay in the presence or absence of recombinant CSFs. For each CSF, the number of colonies is a function of circadian time of bone marrow exposure (ANOVA and Cosinor; p < 0.0001) with the values at peak time being double those found at the trough. Peak CSF efficacy occurred at 3 hours after light onset (HALO, early rest span) irrespective of CSF type or dose. Furthermore, in the absence of any added CSF, the number of clusters varied significantly according to sampling time, with a similar peak at 3 HALO (ANOVA and Cosinor; p < 0.001). Further in vivo chronopharmacologic experiments are needed to assess the relevance of these in vitro rhythms in bone marrow responsiveness to hematopoietic growth factors.

Cellular proliferation in the spleen of DBA/2 mice infected with a myeloproliferative sarcoma virus(MPSV).

Cellular proliferation kinetics were investigated in the spleens of DBA/2 mice infected with a myeloproliferative sarcoma virus (MPSV) at three distinct phases of spleen growth: the rapid-growth phase, the slow-growth phase and the regression phase. Using injection of tritiated thymidine in vivo and autoradiographic techniques, we showed that most blast cells proliferate rapidly (cell cycle is equal to 10 h) even during the slow-growth phase. Very massive and rapid cell loss was found during the rapid-growth phase (cell loss factor phi = 80%). By comparing the decrease in specific splenic activity after in vivo injection of 125IUdR and 3HTdR, we were able to show a large reutilization of tritiated thymidine (R = 57%), visible less than 7 h after injection of isotopic DNA precursors. Thus, MPSV-infected spleen cells were shown, for the most part, to be short-lived cells: almost one-half mature into erythrocytes and the others rapidly die in situ.

20 alpha-hydroxysteroid dehydrogenase expression in a murine virus-induced myeloproliferative syndrome.

The myeloproliferative sarcoma virus (MPSV) infection in DBA/2 mice leads to important quantitative and qualitative changes in their hemopoiesis. These findings suggest a disturbance in the production and action of a certain hemopoietic factor similar to IL3. Here, we show that the level of the 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) expression, which can be induced by IL3, is dramatically increased in spleen and thymus of MPSV-infected mice. Our results suggest that quantification of 20 alpha-SDH activity can be used to indicate abnormal production of a growth factor similar to IL3 in hemopoietic system diseases.

Autocrine growth of leukemic cells.

Autocrine growth is a process whereby a cell both secretes and responds to a growth factor. This paper describes the stepwise malignant progression of leukemic cells which has been demonstrated in many experimental models of autocrine leukemic growth. In contrast, autocrine growth has not been proven as a major physiopathological mechanism for the growth of leukemic cells in vivo in human myeloid and lymphocytic leukemias. Growth-factor independency of human leukemic cell lines may be due to clonal selection.

The role of growth-factor receptors (excluding IL-2 receptors) in the proliferation and differentiation of normal and leukemic hematopoietic cells.

Receptors (R) are considered as allosteric enzymes whose action on metabolic chains is modulated through binding to the ligand. They play an essential role in the transduction of the multiple signals (e.g. interleukins or CSF) which intervene in the regulation of hematopoiesis. Their ordered interactions are necessary to regulate the growth and differentiation of normal hematopoietic precursors. This paper summarizes recent data concerning the structure-action relationship of growth-factor receptors in the signal transduction and alterations of growth-factor receptors which may play an important role in leukemic transformation. Some therapeutic modulations of growth-factors cascades are also discussed.

Isolation from the spleen of myeloproliferative sarcoma virus-infected mice of a myelomonocytic tumor cell line secreting hematopoietic colony stimulating factors.

Clonogenic tumor cells were detected in the spleen of DBA/2 mice infected with the myeloproliferative sarcoma virus (MPSV). These cells could be isolated because of their ability to proliferate on the greater omentum of sublethally irradiated isogenic recipient mice. We have established a permanent suspension myelomonocytic cell line in vitro (TE8), from a tumor obtained after subcutaneous transplantation of the omental tumor masses. The TE8 cells are clonogenic in semi-solid medium containing agarose. The colonies thus obtained gave rise to myelomonocytic cell lines growing in suspension in liquid medium. One of those cloned cell lines, C1(1011) was studied in details. It is exclusively composed of initial donor cells, it is tumorigenic in vivo, clonogenic in vitro and produces MPSV. It also secretes a colony stimulating activity (CSA), and an activity termed mixed colony promoting activity (MPA), which enables pluripotent hematopoietic stem cells to proliferate and differentiate. It differentiates through the granulomacrophage cell line independently of exogenous factors other than those which may be present in the serum.

Abnormal splenic megakaryopoiesis in MPSV-induced myeloproliferative disease.

The myeloproliferative sarcoma virus (MPSV) induces a murine myeloproliferative syndrome characterized by an erythromyelemia, an anemia, a thrombocytopenia associated with a myeloproliferation in the spleen and a splenic and medullar fibrosis. We have used the in-vitro plasma clot technique to measure megakaryocytic precursors in the spleen and bone-marrow of MPSV-infected mice. We report that megakaryocytic colonies are increased, in number (X75), in concentration (X9) and in size, in the spleen but not in the bone-marrow of neoplastic mice. Furthermore, these splenic precursors are hypersensitive to growth factors present in the anemic mouse serum used in the culture system. These data show that the thrombocytopenia observed in the MPSV-induced neoplasia does not result from a lack of megakaryocyte precursors, but rather from an excess of megakaryocyte destruction. This ineffective splenic megakaryopoiesis associated with the presence of a massive splenic fibrosis make the MPS-induced neoplasia a suitable model for studying the perturbation of megakaryopoiesis in myeloproliferative syndrome associated with fibrosis.

In vitro induction of CFU-S proliferation by a non viral splenic activity from myeloproliferative sarcoma virus infected mice.

The myeloproliferative sarcoma virus (MPSV) induces a myeloproliferative syndrome in DBA/2 mice. It is characterized by a considerable increase in the number (100-fold) and in the concentration (10-fold) of pluripotent hematopoietic stem cells detected in vivo (CFU-S) in the spleens of infected animals. Prior studies have shown the presence of a mixed-colony promoting activity (MPA) in neoplastic spleens. In the presence of a small quantity of erythropoietin, MPA induces the proliferation and differentiation of pluripotent hematopoietic stem cells, detected in vitro (Mix-CFU). We tested the effect of factors produced by neoplastic spleen cells on the proliferation of day 10 CFU-S and their entry into the cell cycle. This was done by comparing the number of day 10 CFU-S present in suspensions of normal bone marrow cells incubated for 2 days on agar underlays containing cells from either normal or neoplastic spleens. Our results show the existence of an activity secreted by cells from the spleens of MPSV-infected animals which starts CFU-S cycling and which is physically distinct from MPSV. The presence of this activity, whose identity with MPA remains to be proven, would enable us to explain the proliferation of CFU-S in the course of the disease.

Myeloproliferative Sarcoma Virus stimulates pluripotent hematopoietic stem cells and provokes tumoral transformation of the hematopoietic microenvironment in vitro.

The Myeloproliferative Sarcoma Virus (MPSV) induces an increase in the number and concentration of pluripotent stem cells in long-term murine bone marrow cultures. This is followed by an increased number of precursor cells of the granulocyte and macrophage lines (GM-CFC). This increase is comparable to that observed in DBA/2 mouse spleens in vivo two to three weeks after viral infection. Proliferation of CFUs and GM-CFC decreases five weeks after infection with MPSV, in parallel to the gradual decline of reverse transcriptase activity in the culture medium. GM-CFC which can proliferate in the absence of added colony stimulating factor (CSF) were detected at week 6 post MPSV infection. Adherent tumor cells were observed nine weeks after infection. These fibroblast type cells gave rise to a permanent line which produced a CSF-like activity. Our results show that MPSV causes the tumoral transformation of fibroblast-like cells of the bone-marrow hematopoietic microenvironment. In addition, MPSV also strongly stimulates the proliferation of hematopoietic stem cells. MPSV is, until now, the first murine retrovirus which exhibits such properties.

CD44 and hyaluronan binding by human myeloid cells.

The CD44 cell surface molecule has been shown to be the principal cell surface receptor for hyaluronan (or hyaluronic acid), a glycosaminoglycan component of marrow extracellular matrix. However, its affinity for hyaluronan is not constitutive, since it depends on the cell type, the stage of differentiation and on activation by external stimuli including certain anti-CD44 antibodies and phorbol esters. Except for a few lymphoid cell lines, hematopoietic cells do not spontaneously bind hyaluronan and initial studies reported that, contrary to lymphocytes, myeloid cells could not be activated to bind hyaluronan. Because CD44 plays an important role in the initial phases of hematopoiesis, as shown by experiments using blocking anti-CD44 monoclonal antibodies, its capacity to mediate adhesion of primitive myeloid cells has been investigated. It was found that CD44 could mediate spontaneous adhesion to hyaluronan of immature myeloid cell lines KG1, KG1a, and TF1, which serve as a model for hematopoietic progenitors. However, despite expressing high amounts of CD44, no more than 15% of bone marrow progenitors could adhere to hyaluronan. Recent experiments have shown that a very important feature of CD44 is its capacity to be rapidly activated by certain antibodies and cytokines (GM-CSF and KL) from a low affinity to a high affinity state for hyaluronan. These data shed light on striking similarities in the functional regulation of CD44 and of the two integrin receptors VLA-4 (a4b1), and VLA-5 (a5b1), which are also expressed on hematopoietic progenitors. The relevance of these data to the regulation of normal hematopoiesis and mobilization of CD34+ progenitors in the view of cell grafting is analyzed. In addition, we show that in idiopathic myelofibrosis, the amount of hyaluronan is markedly increased in the extracellular matrix from the myeloproliferative spleen. Considering that the production of cytokines is enhanced in this disease, we discuss whether CD44-hyaluronan interaction may have a role in the pathophysiology of this myeloproliferative syndrome.

Histopathologic studies on myeloproliferative sarcoma virus (MPSV) induced leukemias and hemangiosarcoma in Jar-2 rats.

It is well known that MPSV induces myeloproliferative syndrome (MPS) in mice. Intravenous one shot inoculation of myeloproliferative sarcoma virus (MPSV) with Friend murine leukemia virus (F-MuLV) as a helper in newborn Jar-2 rats (on the second neonatal day) yielded hematopoietic malignancies in all the treated rats (25/25 rats) after 2 weeks' latency. MPS appeared from the 14th day in 14 rats. In the midst of the myeloproliferative field of the spleen and bone marrow, myeloblastic or myeloblastic-erythroblastic foci were observed. From 19th day, acute myeloblastic leukemia occurred in 3 rats and erythroleukemia in 8 rats. MPSV induced first MPS which remained as such or later developed into acute leukemia. Myelofibrosis as seen in mice was not observed. In addition, hemangiosarcoma of the brain, spinal cord and spleen appeared in 15 rats from the 24th day, and were often multiple. MPSV can yield the tumor only in newborn rats, and target cells of MPSV are not only hematopoietic cells but also endothelial cells of the brain, spinal cord and occasionally spleen.