Robert Hromas

The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1.

EWS/FLI-1 is a chimeric protein formed by a tumor-specific 11;22 translocation found in both Ewings sarcoma and primitive neuroectodermal tumor of childhood. EWS/FLI-1 has been shown to be a potent transforming gene, suggesting that it plays an important role in the genesis of these human tumors. We now demonstrate that EWS/FLI-1 has the characteristics of an aberrant transcription factor. Subcellular fractionation experiments localized the EWS/FLI-1 protein to the nucleus of primitive neuroectodermal tumor cells. EWS/FLI-1 specifically bound in vitro an ets-2 consensus sequence similarly to normal FLI-1. When coupled to a GAL4 DNA-binding domain, the amino-terminal EWS/FLI-1 region was a much more potent transcriptional activator than the corresponding amino-terminal domain of FLI-1. Finally, EWS/FLI-1 efficiently transformed NIH 3T3 cells, but FLI-1 did not. These data suggest that EWS/FLI-1, functioning as a transcription factor, leads to a phenotype dramatically different from that of cells expressing FLI-1. EWS/FLI-1 could disrupt normal growth and differentiation either by more efficiently activating FLI-1 target genes or by inappropriately modulating genes normally not responsive to FLI-1.

Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

PLAB, a novel placental bone morphogenetic protein

Bone morphogenetic proteins (BMP) constitute a sub-group of the large transforming growth factor-beta (TGF-beta) family. They play important roles in the embryonic development of multiple structures and in adult bone modeling. We have recently isolated a novel member of the BMP family from placenta, termed PLAB. PLAB is expressed highly in placenta, but can be found upon stringent analysis in low levels in most other tissues. At the amino acid level, PLAB is most closely related to BMP-8/OP-2, another member of the BMP family. Like TGF-beta, PLAB inhibits the proliferation of primitive hematopoietic progenitors. The high expression of PLAB by placenta raises the possibility that it may be a mediator of placental control of embryonic development.

The embryonic stem cell transcription factors Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression

The POU homeodomain protein Oct-4 and the Forkhead Box protein FoxD3 (previously Genesis) are transcriptional regulators expressed in embryonic stem cells. Down-regulation of Oct-4 during gastrulation is essential for proper endoderm development. After gastrulation, FoxD3 is generally down-regulated during early endoderm formation, although it specifically remains expressed in the embryonic neural crest. In these studies, we have found that Oct-4 and FoxD3 can bind to identical regulatory DNA sequences. In addition, Oct-4 physically interacted with the FoxD3 DNA-binding domain. Cotransfection of Oct-4 and FoxD3 expression vectors activated the osteopontin enhancer, which is expressed in totipotent embryonic stem cells. FoxA1 and FoxA2 (previously HNF-3α and HNF-3β) are Forkhead Box transcription factors that participate in liver and lung formation from foregut endoderm. Although FoxD3 activated the FoxA1 and FoxA2 promoters, Oct-4 inhibited FoxD3 activation of the FoxA1 and FoxA2 endodermal promoters. These data indicate that Oct-4 functions as a corepressor of FoxD3 to provide embryonic lineage-specific transcriptional regulatory activity to maintain appropriate developmental timing.

Chemokine Regulation of Normal and Pathologic Immune Responses

Chemokines are small basic proteins that are the major mediators of all leukocyte migration. There are at least 46 distinct chemokines, and 19 chemokine receptors, making it easily the largest cytokine family. Chemokines can be both beneficial and harmful, by either stimulating an appropriate immune response to microbial invasion, or by mediating pathologic tissue destruction in many types of human disease. Chemokines have been implicated in the tissue destruction seen in autoimmune diseases, atherosclerosis, allograft rejection, and neoplasia. Chemokines also play essential roles in normal lymphocyte trafficking to primary and secondary lymphoid organs for antigen presentation and lymphocyte maturation. Chemokines also regulate hematopoietic stem and progenitor cell homing and proliferation. Therefore, it is likely that chemokines will become important targets for pharmacologic intervention in a wide variety of human diseases in the future.

Effects of CC, CXC, C, and CX3C Chemokines on Proliferation of Myeloid Progenitor Cells, and Insights into SDF-1-Induced Chemotaxis of Progenitorsa

Abstract: Chemokines have been implicated in the regulation of stem/progenitor cell proliferation and movement. The purpose of the present study was to assess a number of new chemokines for suppressive activity and to delve further into SDF‐1‐mediated chemotaxis of progenitor cells. This report extends the list of chemokines that have suppressive activity against immature subsets of myeloid progenitors stimulated to proliferate by multiple growth factors to include: MCP‐4/CKβ‐10, MIP‐4/CKβ‐7, I‐309, TECK, GCP‐2, MIG and lymphotactin. The suppressive activity of a number of other chemokines was confirmed. Additionally, pretreatment of the active chemokines with an acetylnitrile solution enhanced specific activity of a number of these chemokines. The new chemokines found to be lacking suppressive activity include: MCP‐2, MCP‐3, eotaxin‐1, MCIF/HCC‐1/CKβ‐1, TARC, MDC, MPIF‐2/eotaxin‐2/CKβ‐6, SDF‐1 and fractalkine/neurotactin. Overall, 19 chemokines, crossing the CC, CXC, and C subgroups, have now been found to be myelosuppressive, and 14 chemokines crossing the CC, CXC and CX3C subgroups have been found to lack myelosuppressive activity under the culture conditions of our assays. Because of the redundancy in chemokine/chemokine receptor interactions, it is not yet clear through which chemokine receptors many of these chemokines signal to elicit suppressive activities. It was also found that SDF‐1‐induced chemotaxis of progenitors can occur in the presence of fibronectin (FN) and extracellular matrix components and that FN effects involve activation of β1‐, and possibly α4‐, integrins.

Hematopoietic transcriptional regulation by the myeloid zinc finger gene, MZF-1.

Transcriptional regulators control much of hematopoiesis. One such transcriptional regulator is the myeloid zinc finger gene MZF-1. MZF-1 has been localized to the telomere of chromosome 19q, where a large number of related zinc finger genes reside. It has been found to be essential in granulopoiesis. It is a bi-functional transcriptional regulator, repressing transcription in non-hematopoietic cells, and activating transcription in cells of hematopoietic origins. Its consensus DNA binding site has been isolated, and sites in several promoters of myeloid-specific genes, such as CD34, lactoferrin, and myeloperoxidase, have been defined. In co-transfection experiments MZF-1 has been found to regulate transcription from the CD34 promoter.

Tandem high dose chemotherapy with autologous bone marrow transplantation for initial relapse of testicular germ cell cancer

The purpose of this study was to evaluate the use of two cycles of high dose chemotherapy with autologous bone marrow transplantation (ABMT) in the treatment of patients having a first relapse of testicular germ cell cancer.

The promyelocytic leukemia protein PML interacts with the proline-rich homeodomain protein PRH: a RING may link hematopoiesis and growth control

Acute promyelocytic leukemia (APL) is characterized by a block in myeloid cell differentiation. As a result of a chromosomal translocation in these patients, the promyelocytic leukemia protein PML is disrupted as are the nuclear bodies it forms. Disruption of PML and PML nuclear bodies in APL is linked to a loss of growth control and subsequent leukemogenesis. PML contains a zinc-binding domain known as the RING which is required for formation of these bodies. Using yeast 2-hybrid techniques, we found that PML and a related RING protein, Z, bind the proline rich homeodomain protein (PRH) through their RING domains. Previous reports indicate that PRH functions in hematopoiesis and may act as a transcriptional repressor. Our data indicate that PML and Z both bind the repressor domain of PRH and are the first protein partners reported for PRH. We observe that PRH has a punctate pattern in both the nucleus and cytoplasm of chronic myelogenous leukemia K562 cells and in the APL cell line, NB4. Immunoprecipitation and co-localization studies indicate that PML and PRH interact in both cell lines. The effect on cell growth by PML and the hematopoietic actions of PRH raises the possibility that the interaction between PML and PRH represents a link between growth control and hematopoiesis.

Genesis, a Winged Helix transcriptional repressor, has embryonic expression limited to the neural crest, and stimulates proliferation in vitro in a neural development model

Abstract A novel repressor of the Winged Helix (formerly HNF-3/Forkhead) transcriptional regulatory family, termed Genesis (also called HFH2), was previously found to be exclusively expressed in primitive embryonic cell lines. In this study in situ cRNA hybridization experiments revealed that Genesis was expressed during embryogenesis only in developing neural crest cells. Its expression diminished upon their terminal differentiation into sympathetic and parasympathetic neurons. Based on that finding, Genesis was retrovirally transduced into pluripotent N-Tera-2 clone D1 (NT2/D1) teratocarcinoma cells, which are a well-described in vitro model of neural development. Retinoic acid (RA) treatment will drive these cells to differentiation toward the neuronal lineage and cause an increase in expression of the cyclin-dependent kinase inhibitor p21 protein, which leads to an inhibition in cellular proliferation. Although RA-induced expression of neuronal differentiation markers was not influenced by forced overexpression of Genesis in NT2-D1 cells, proliferation of Genesis-transduced cells continued following RA treatment. RA was unable to induce the expression of the cyclin-dependent kinase inhibitor p21 in the Genesis-transduced cells, but Go/G1 tumor suppressor p53 expression was induced normally. Therefore, Genesis may play a role in the regulation of primitive neural crest development by preventing terminal quiescence through inhibition of p21 protein expression. These data also lend evidence for the hypothesis that proliferation and differentiation pathways are not irrevocably linked, but can function independently.