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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-3alpha and HNF-3beta) 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.

Between October 1991 and May 1994, 42 patients were treated with cyclophosphamide, thiotepa, and total body irradiation followed by an allogeneic transplantation of marrow depleted of T cells with soybean agglutinin and E-rosetting. Patients included in this study had acute myelogenous leukemia (13), chronic myelogenous leukemia (12), acute lymphocytic leukemia (nine), Hodgkin's disease or non-Hodgkin's lymphoma (four), multiple myeloma (three), or myelodysplastic syndrome (one). The mean age was 34 (range 8 to 51 years). Nineteen patients had a matched sibling donor and 18 received marrow from 6/6 matched unrelated donors while five received transplants from unrelated donors disparate at one DR locus (5/6 match). Time to granulocyte engraftment (AGCor = 500/mm3) occurred at a mean of 16.5 days for related and 11.4 days for unrelated transplant recipients, and was related to the increased use of G-CSF in the unrelated population. There was no correlation with number of mononuclear cells, T cells, or CD34-positive cells infused, the rate of engraftment or the incidence of transplant complications. Multivariate analysis determined that G-CSF administration and a diagnosis other than ALL were the only factors associated with a faster rate of engraftment. Patients receiving unrelated donor transplants, those with ALL, or those who had a low T cell number infused (or = 8.0 x 10(3) cells/kg) experienced delayed hospital discharge. The regimen resulted in excellent rates of engraftment (95.2%) with only one failure to engraft and one graft rejection. The incidence of grade III-IV acute graft-versus-host disease was 0% with sibling and 26.1% with unrelated donors. There were no cases of veno-occlusive disease. Fifty percent of patients are alive with a mean follow-up of 26.4 months. We conclude that this regimen is well tolerated and results in excellent engraftment with a low incidence of severe graft-versus-host disease and few therapy-related toxicities.

The myeloid zinc finger protein MZF-1 is important in hematopoiesis. Previous studies have found that reducing expression of MZF-1 inhibited G-CSF-driven human marrow colony formation assays. In this study we found that retrovirally overexpressing MZF-1 in IL-3-dependent FDCP.1 cells inhibited their apoptosis when IL-3 was withdrawn. The MZF-1-transduced FDCP.1 cells also formed tumors when injected into congenic mice, whereas control FDCP.1 cells did not.

MZF-1 is a C2H2 zinc finger gene encoding a putative transcriptional regulator of myeloid differentiation. The MZF-1 protein contains 13 C2H2 zinc fingers arranged in bipartite DNA binding domains containing zinc fingers through 4 and, in the carboxy-terminus, 5 through 13. We previously identified the DNA consensus binding site recognized by the two DNA binding domains. To assess the transcription regulatory function of MZF-1, the full-length MZF-1 coding region was fused to the DNA binding domain of the yeast transactivator GAL4. The expression vector was cotransfected with the chloramphenicol acetyl transferase (CAT) reporter gene regulated by the thymidine kinase promoter containing GAL4 DNA binding sites into NIH 3T3, 293, K562, and Jurkat cell lines. MZF-1 represses CAT reporter gene expression via GAL4 binding sites in the nonhematopoietic cell lines NIH 3T3 and 293. In contrast, MZF-1 activates CAT reporter gene expression in the hematopoietic cell lines K562 and Jurkat. The MZF-1 binding sites are present in the promoters of several genes expressed during myeloid differentiation, including the CD34 promoter. MZF-1 transcriptional regulation of this physiologically relevant promoter was assessed in both hematopoietic and nonhematopoietic cell lines. Recombinant MZF-1 protein specifically binds to the consensus binding sites in the CD34 promoter in mobility shift assays. MZF-1 expression vectors were cotransfected with the luciferase reporter plasmids regulated by the CD34 promoter into both nonhematopoietic and hematopoietic cell lines. As with the heterologous DNA binding domain, MZF-1 represses reporter gene expression in nonhematopoietic cell lines and activates expression in hematopoietic cell lines. Activation of CD34 expression in hematopoietic cell lines is dependent on the presence of intact MZF-1 binding sites. The cell type-specific regulation of the CD34 promoter by MZF-1 suggests the presence of tissue-specific regulators/adapters or differential MZF-1 modifications that determine MZF-1 transcriptional regulatory function.

A 29-year-old woman underwent a T cell-depleted unrelated donor transplant for CML in chronic phase. Sixty-three days after marrow infusion, the patient developed fevers and generalized lymphadenopathy. Lymph node biopsy was consistent with monoclonal EBV-associated immunoblastic lymphoma for which the patient received 10(5) CD3-positive donor leukocytes per kilogram. Six days after leukocyte infusion the patient developed mental status changes without focal neurological deficit. MRI revealed no mass lesions. Cerebral spinal fluid revealed a white blood cell count of 1650 cells/mm3 which were shown to be T lymphocytes of donor origin. The CSF was tested and found to be PCR positive for EBV virus interval repeat 1 sequence (IR1). The lymphocytosis and mental status changes resolved without specific intervention. Subsequently she developed marrow aplasia, which was believed to be secondary to the infusion of donor leukocytes. Possible mechanisms for these two previously unreported side-effects of donor leukocyte infusion are discussed.

Thirty-three patients with germ cell cancer (GCT) recurrent after two cisplatin-based regimens or cisplatin refractory (progression within 4 weeks of the last dose of cisplatin) were enrolled in a trial to establish the maximum tolerated doses (MTD) of carboplatin and etoposide given in combination with ABMT for two cycles. BM harvest ofor = 2 x 10(8) nucleated cells/kg preceded two cycles of therapy. Each agent was dose escalated, carboplatin from 1650 mg/m2 to 2100 mg/m2 and etoposide from 1200 mg/m2 to 2250 mg/m2 per cycle in successive cohorts. Twenty patients completed two cycles, 13 underwent only one due to: early death (4), toxicity (2), and progressive disease (6). There were four CR, three of whom achieved NED status with surgery, 14 PR, of whom eight have progressed. Four patients with stable disease and seven PD have died with a median survival of 6 months. There were six treatment-related deaths, four on course 1 and two on course 2. Causes of death on course 1 were: CNS hemorrhage (1), multiorgan failure (3); and on course 2: sepsis (1) and sudden death (1). Severe but reversible mucositis, transaminase and creatinine elevations were observed at the highest dose level. Three of five patients treated at this dose level had severe neurologic toxicity, manifested by both peripheral neuropathy and ototoxicity. The MTD in this patient population was carboplatin 2100 mg/m2 and etoposide 2250 mg/m2 on each of two cycles of therapy. Neurologic and mucosal toxicity were dose limiting.

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.

The zinc finger gene MZF-1 is preferentially expressed in primitive hematopoietic cells and plays an important role in regulating myelopoiesis. Regulators of development are potential targets for neoplastic transformation. This study investigated whether unregulated expression of MZF-1 could function as an oncogene. Retroviral transduction and subsequent overexpression of MZF-1 resulted in loss of contact inhibition, loss of substrate dependence, and more rapid cell cycling in NIH 3T3 cells. The MZF-1-transformed 3T3 cells formed aggressive tumors in athymic mice. Disruption of the tight lineage- and stage-specific regulation of MZF-1 can result in neoplastic transformation of embryonic fibroblasts. Therefore, MZF-1 represents a novel oncogene.