A bcr-3 isoform of RARalpha-PML potentiates the development of PML-RARalpha-driven acute promyelocytic leukemia.

Acute promyelocytic leukemia (APML) most often is associated with the balanced reciprocal translocation t(15;17) (q22;q11.2) and the expression of both the PML-RARalpha and RARalpha-PML fusion cDNAs that are formed by this translocation. In this report, we investigated the biological role of a bcr-3 isoform of RARalpha-PML for the development of APML in a transgenic mouse model. Expression of RARalpha-PML alone in the early myeloid cells of transgenic mice did not alter myeloid development or cause APML, but its expression significantly increased the penetrance of APML in mice expressing a bcr-1 isoform of PML-RARalpha (15% of animals developed APML with PML-RARalpha alone vs. 57% with both transgenes, P < 0.001). The latency of APML development was not altered substantially by the expression of RARalpha-PML, suggesting that it does not behave as a classical "second hit" for development of the disease. Leukemias that arose from doubly transgenic mice were less mature than those from PML-RARalpha transgenic mice, but they both responded to all-trans retinoic acid in vitro. These findings suggest that PML-RARalpha drives the development of APML and defines its basic phenotype, whereas RARalpha-PML potentiates this phenotype via mechanisms that are not yet understood.

Altered myeloid development and acute leukemia in transgenic mice expressing PML-RAR alpha under control of cathepsin G regulatory sequences.

Acute promyelocytic leukemia (APML) is characterized by abnormal myeloid development, resulting an accumulation of leukemic promyelocytes that are often highly sensitive to retinoic acid. A balanced t(15;17) (q22;q21) reciprocal chromosomal translocation is found in approximately 90% of APML patients; this translocation fuses the PML gene on chromosome 15 to the retinoic acid receptor alpha (RAR alpha) gene on chromosome 17, creating two novel fusion genes, PML-RAR alpha and RAR alpha-PML. The PML-RAR alpha fusion gene product, which is expressed in virtually all patients with t(15;17), is thought to play a direct role in the pathogenesis of APML. To determine whether PML-RAR alpha is sufficient to cause APML in an animal model, we used the promyelocyte-specific targeting sequences of the human cathepsin G (hCG) gene to direct the expression of a PML-RAR alpha cDNA to the early myeloid cells of transgenic mice. Mice expressing the hCG-PML-RAR alpha transgene were found to have altered myeloid development that was characterized by increased percentages of immature and mature myeloid cells in the peripheral blood, bone marrow, and spleen. In addition, approximately 30% of transgene-expressing mice eventually developed acute myeloid leukemia after a long latent period. The splenic promyelocytes of mice with both the nonleukemic and leukemic phenotypes responded to all-trans retinoic acid (ATRA) treatment, which caused apoptosis of myeloid precursors. Although low-level expression of the hCG-PML-RAR alpha transgene is not sufficient to directly cause acute myeloid leukemia in mice, its expression alters myeloid development, resulting in an accumulation of myeloid precursors that may be susceptible to cooperative transforming events.

Regulation of azurophil granule-associated serine protease genes during myelopoiesis.

During myelopoiesis, the primary or azurophil granules are synthesized at the promyelocyte stage of development. The biogenesis of these granules requires the coordinated regulation of a large number of genes that comprise the constituent proteins of the granules themselves. The genes encoding the granule-associated proteins appear to be regulated primarily at the level of gene transcription; that is, most of the granule genes appear to be transcriptionally activated at the beginning of the promyelocyte stage and are transcriptionally repressed at the transition to myelocytes. The cis-acting DNA elements and trans-regulatory factors that control the regulation of granule genes are just beginning to be defined. The granule-associated genes are dispersed throughout the genome; however, several of the serine protease genes expressed in these granules are tightly clustered and the tight clustering may play an important role for their regulation. Recent transgenic studies suggest that DNA elements required for myeloid-specific targeting may be located just upstream from or very near to the granule genes themselves. DNA elements similar to the globin locus control region will probably play a role in the expression of these genes, although locus control elements have not yet been defined for any azurophil granule genes. Future studies directed at defining these key regulatory elements will enhance our understanding of the molecular events that underlie myeloid development.

Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice.

The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-specific "targeting," we injected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, approximately 2.5 kb of 5' flanking sequence, and approximately 0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable transgenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of transgenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myeloid cells. These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment.

Consensus AP-1 and CRE motifs upstream from the human cytotoxic serine protease B (CSP-B/CGL-1) gene synergize to activate transcription.

The human CGL-1/cytotoxic serine protease B gene (CSP-B; also known as granzyme B) is transcriptionally activated during cytotoxic T-lymphocyte maturation. Activation can be mimicked in the PEER T-cell leukemia cell line by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (bt2cAMP). In this report, we show that a consensus AP-1 element and a consensus cAMP response element (CRE) located 5' to the CSP-B transcriptional start site are both required for transcriptional activation of the CPS-B promoter in TPA + bt2cAMP-stimulated PEER cells. A 94-bp fragment containing both elements activates a heterologous promoter in an orientation-independent fashion. Several single nucleotide substitutions in the AP-1 site abolish activity of the 94-bp fragment. Several point mutations in the consensus CRE substantially reduce promoter activity, but one CRE mutation increases activity fourfold. Replacement of the CRE with a second copy of the AP-1 site results in a level of transcriptional activity comparable with that of the wild-type sequence, but replacement of the AP-1 site with a CRE abolishes activity. Neither the AP-1 site nor the CRE can be effectively replaced with an SP-1 site. Deletions between the AP-1 site and the CRE retain full activity only if helical spacing is preserved, suggesting that synergism between these two elements is either the result of cooperative binding of factors to the DNA or of cooperative binding of DNA-bound factors to another protein.

Magnetic resonance imaging and intraocular foreign bodies.

We conducted in vitro and in vivo experiments to study magnetic resonance imaging of intraocular foreign bodies. Diamagnetic and paramagnetic foreign bodies were imaged without artifact and without movement during the imaging process, while ferromagnetic foreign bodies produced large amounts of artifact that prevented meaningful images. All fetromagnetic foreign bodies moved during in vitro imaging. During in vivo imaging, three of four ferromagnetic bodies moved, producing substantial retinal injury. We concluded that magnetic resonance imaging is contraindicated in traumatized eyes with suspected ferromagnetic foreign bodies.

Progressive hemifacial atrophy (Parry-Romberg disease).

Hemifacial atrophy (Parry-Romberg syndrome) is characterized by slowly progressive atrophy of one side of the face, primarily involving the subcutaneous tissue and fat. The onset is usually in the first two decades. Ophthalmic involvement is common; the most frequent abnormality is progressive endophthalmos with subsequent changes in the palpebral fissure. Pupillary disturbances, heterochromia, uveitis, and restrictive strabismus have also been frequently reported. We describe six cases that manifest a wide spectrum of ocular and systemic findings. They are noteworthy in that all exhibit pigmentary disturbances of the ocular fundus, a finding rarely reported. Another unusual ocular manifestation in one patient was an acquired partial third nerve palsy on the unaffected side.

An automatic laser filter for the indirect ophthalmoscope.

We have developed an automatic filter for the indirect ophthalmoscope that protects the physician's eye from potential damage during laser treatment. The laser light is applied through an endolaser probe using indirect ophthalmoscopy.

Technique for trouble-free removal of pars plana infusion cannulas.

We describe a simple procedure that facilitates the removal of a pars plana cannula. A 2-cm 4-O black silk suture is passed under the double-throw knot, which secures the infusion cannula. When the ends of the silk suture are pulled, the double-throw knot opens easily, allowing the cannula to be removed.

Malignant uveal melanoma and simulating lesions: MR imaging evaluation.

Twenty-one patients with intraocular disease were studied by magnetic resonance (MR) imaging and computed tomography (CT). In 13 cases, malignant uveal melanoma was considered the likely diagnosis. Both imaging methods were accurate in determining the location and size of uveal melanomas. MR imaging was superior for the assessment of possible associated retinal detachment, for assessment of vitreous change, and for differentiating uveal melanoma from choroidal hemangioma and choroidal detachment. A case of retinal gliosis could not be differentiated from uveal melanoma by either technique. Uveal melanomas appeared as hyperintense lesions on T1-weighted images and as hypointense lesions on T2-weighted images. High signal intensity of the vitreous was observed in patients with vitritis and in those who were thought to have protein leaking into the vitreous as a result of impairment of the retinal-blood barrier.

Special short needles to inject and aspirate high-viscosity silicone oil.

The use of 16-gauge needles of shortened length (4 and 8 mm) allows intraocular injection of high-viscosity silicone oil (12,500 centistokes) without the need for special pumps.

Retinal toxicity study of intravitreal cyclosporin.

We investigated the toxicity of intravitreal cyclosporin in the rabbit. Histologic retinal changes were found with intravitreal injections of 200 micrograms. No histologic or electroretinographic changes were observed with doses of 100 micrograms or less. This nontoxic dose is projected to be at least 77 times the amount needed to be effective.

Intraocular photocoagulation with the argon-krypton laser.

We have developed an intraocular laser coagulator using a low-loss single-fiber probe system connected to an argon-krypton laser. Coagulation can be accomplished at distances of 3 mm or greater from the retina, permitting easy control of lesion size, location, and intensity. The probe can be fired rapidly without overheating and can also be fired into air without damage to the tip.