Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-alpha bigenic mice

Overexpression of ErbB-2/Neu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)/Neu + MMTV/transforming growth factor alpha bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogen-activated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27(Kip1). In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1/2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 and MAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478-treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.     

"Stem cell" origin of the hematopoietic defect in dyskeratosis congenita

We have used the long-term bone marrow culture (LTBMC) system to analyze hematopoiesis in three patients with dyskeratosis congenita (DC), two of whom had aplastic anemia, and the third had a normal blood count (apart from mild macrocytosis) and normal BM cellularity. Hematopoiesis was severely defective in all three patients, as measured by a low incidence of colony-forming cells and a low level of hematopoiesis in LTBMC. The function of the marrow stroma was normal in its ability to support the growth of hematopoietic progenitors from normal marrows seeded onto them in all three cases, but the generation of hematopoietic progenitors from patients marrow cells inoculated onto normal stromas was reduced, thus suggesting the defect to be of stem cell origin. The parents and unaffected brother of one of the families have also been studied in LTBMC and all showed normal hematopoietic and stromal cell function. From this study we speculate that there are some similarities between DC and the defect in the W/Wv mouse.     

Plasma levels of plasminogen activator inhibitor type 1, beta- thromboglobulin, and fibrinopeptide A before, during, and after treatment of acute myocardial infarction with alteplase

Plasma levels of plasminogen activator inhibitor type-1 (PAI-1), beta- thromboglobulin (beta TG), and fibrinopeptide A (FPA) were followed over 24 hours in 30 patients treated with alteplase for acute myocardial infarction. Samples were taken at baseline (T Oh), after 90 minutes (under alteplase, no heparin, T 1.5h), after 120 minutes (under alteplase and heparin, T 2h), 30 minutes after thrombolytic therapy (T 3.5h), as well as 12 hours (T 12h) and 24 hours (T 24h) after baseline. PAI-1 antigen levels (55 +/- 9 ng/mL at T Oh, mean +/- SEM) decreased to 35 +/- 5 (T 1.5h) and 40 +/- 6 (T 2h) ng/mL under alteplase, before increasing to 84 +/- 22 (T 3.5h), 130 +/- 30 (T 12h), and 64 +/- 7 (T 24h) ng/mL after therapy, P less than .001. A high baseline PAI-1 activity (18 +/- 3 ng/mL) decreased to 2.0 +/- 0.4 (T 1.5h) and 1.7 +/- 0.2 (T 2h) under alteplase and increased to 32 +/- 5 (T 12h) and 19 +/- 3 (T 24h) ng/mL after therapy (P less than .0001). beta TG levels (339 +/- 105 ng/mL at T Oh) decreased to 203 +/- 48 (T 2h), 154 +/- 51 (T 3.5h), 187 +/- 40 (T 12h), and 142 +/- 32 (T 24h) ng/mL under heparin (P less than .01). FPA levels (34 +/- 9 ng/mL at T Oh) increased to 85 +/- 15 ng/mL under alteplase alone (T 1.5h) and normalized under heparin (11 +/- 4, 6 +/- 2, 4 +/- 2, and 3 +/- 1 ng/mL at T 2h, T 3.5h, T 12h, and T 24h, respectively). A high level of FPA at T 3.5h correlated with reocclusion (33 +/- 12 ng/mL, n = 4 v 2.9 +/- 0.5 ng/mL, n = 21, P less than .005). We conclude that plasma levels of PAI- 1 antigen as well as activity markedly increase after alteplase therapy of acute myocardial infarction. The high activity of PAI-1 and decreasing beta TG levels suggest that platelets do not contribute significantly to this phenomenon. The marked increase of FPA levels under recombinant tissue-type plasminogen activator alone and its normalization under heparin emphasize the important role of concomitant anticoagulation in controlling further intravasal fibrin generation under alteplase.     

Heterogeneity of B cell involvement in acute nonlymphocytic leukemia

In order to study the pattern of B cell involvement in acute nonlymphocytic leukemia (ANLL), multiple B lymphoid cell lines were established by Epstein-Barr virus transformation of peripheral blood mononuclear cells from two patients with the disease who were heterozygous for the X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD). In one patient, the progenitor cells involved by the leukemia exhibited multipotent differentiative expression, whereas in the other patient the cells showed differentiative expression restricted to the granulocytic pathway. In the patient whose abnormal clone showed multipotent expression, the ratio of B-A G6PD in B lymphoid cell lines was skewed in the direction of type B (the enzyme characteristic of the leukemia clone) and significantly different from the 1:1 ratio expected. It is, therefore, likely that the neoplastic event occurred in a stem cell common to the lymphoid series as well as to the myeloid series. In contrast, evidence for B cell involvement was not detected in the patient whose ANLL progenitor cells exhibited restricted differentiative expression. These findings underscore the heterogeneity of ANLL. Clinically and morphologically similar malignancies in these two patients originated in progenitors with different patterns of stem cell differentiative expression. This difference may reflect differences in cause and pathogenesis.     

The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro

Factors that regulate the growth and development of primitive bone marrow stromal cell precursors are not well defined. We have examined 25 purified recombinant growth factors for their ability to initiate and support clonogenic growth of fibroblast colony-forming cells (CFU- F) from adult human bone marrow. Assays were performed using bone marrow mononuclear cells (BMMNC) enriched in CFU-F by magnetic- activated cell sorting (MACS) using the monoclonal antibody (MoAb) STRO- 1. A serum-deprived assay was developed to avoid components of fetal calf serum (FCS) that may mask or otherwise modify the response of CFU- F to exogenously added factors. L-ascorbate and the glucocorticoid dexamethasone were found to be essential for CFU-F colony development under serum-deprived conditions. Importantly, clonogenic growth of CFU- F in this culture system was absolutely dependent on an exogenous source of growth factor. Platelet-derived growth factor-BB (PDGF) and epidermal growth factor (EGF) demonstrated the greatest ability to support colony growth. Colony formation was dose-dependent, with half- maximal colony numbers at approximately 0.2 ng/mL for either factor and plateau numbers at concentrations in excess of 1.0 ng/mL. Simultaneous addition of PDGF and EGF had no effect on the number of colonies initiated but resulted in dose-dependent increases in mean colony diameter that were significant (P < or = .05) when compared with the effect of either factor alone or with the size of colonies elicited in control cultures by 20% FCS. Fluorescence-activated cell sorting (FACS) of BMMNC using MoAbs to the alpha chain of the PDGF receptor and to the EGF receptor in combination with the Moab STRO-1 demonstrated constitutive expression of both receptors by greater than 90% on CFU-F. Receptors for insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) were also detected on STRO-1+ CFU-F, but in vitro both IGF- 1 and NGF did not support colony growth. This report demonstrates the development of a simple, reproducible, and stringent culture system for the growth and assay of stromal precursors under serum-deprived conditions and represents an important prerequisite for future studies of the role of growth factors in the regulation of stromal cell proliferation, differentiation, and development.     

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1- stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.     

Organization of the gene for human platelet/endothelial cell adhesion molecule-1 shows alternatively spliced isoforms and a functionally complex cytoplasmic domain

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell-cell adhesion molecule that is expressed on circulating platelets, on leukocytes, and at the intercellular junctions of vascular endothelial cells and mediates the interactions of these cells during the process of transendothelial cell migration. The cDNA for PECAM-1 encodes an open reading frame of 738 amino acids (aa) that is organized into a 27- aa signal peptide, a 574-aa extracellular domain composed of 6 Ig homology units, and a relatively long cytoplasmic tail of 118 aa containing multiple sites for posttranslational modification and postreceptor signal transduction. To provide a molecular basis for the precise evaluation of the structure and function of this transmembrane glycoprotein, we have determined the organization of the human PECAM-1 gene. The PECAM-1 gene, which has been localized to human chromosome 17, is a single-copy gene of approximately 65 kb in length and is broken into 16 exons by introns ranging in size from 86 to greater than 12,000 bp in length. Typical of other members of the Ig superfamily, each of the extracellular Ig homology domains is encoded by a separate exon, consistent with PECAM-1 having arisen by gene duplication and exon shuffling of ancestral Ig superfamily genes. However, the cytoplasmic domain was found to be surprisingly complex, being encoded by seven short exons that may represent discrete functional entities. Alternative splicing of the cytoplasmic tail appears to generate multiple PECAM-1 isoforms that may regulate phosphorylation, cytoskeletal association, and affinity modulation of the mature protein. Finally, a processed pseudogene having 76% identity with PECAM- 1 cDNA was identified and localized to human chromosome 3. These findings should have important implications for structure/function analysis of PECAM-1 and its role in vascular adhesive interactions.     

Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that "niches" need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.