Image-guided and passively tumour-targeted polymeric nanomedicines for radiochemotherapy.

Drug targeting systems are nanometer-sized carrier materials designed for improving the biodistribution of systemically applied (chemo-) therapeutics. Reasoning that (I) the temporal and spatial interaction between systemically applied chemotherapy and clinically relevant fractionated radiotherapy is suboptimal, and that (II) drug targeting systems are able to improve the temporal and spatial parameters of this interaction, we have here set out to evaluate the potential of 'carrier-based radiochemotherapy'. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were used as a model drug targeting system, doxorubicin and gemcitabine as model drugs, and the syngeneic and radio- and chemoresistant Dunning AT1 rat prostate carcinoma as a model tumour model. Using magnetic resonance imaging and gamma-scintigraphy, the polymeric drug carriers were first shown to circulate for prolonged periods of time, to localise to tumours both effectively and selectively, and to improve the tumour-directed delivery of low molecular weight agents. Subsequently, they were then shown to interact synergistically with radiotherapy, with radiotherapy increasing the tumour accumulation of the copolymers, and with the copolymers increasing the therapeutic index of radiochemotherapy (both for doxorubicin and for gemcitabine). Based on these findings, and on the fact that its principles are likely broadly applicable, we propose carrier-based radiochemotherapy as a novel concept for treating advanced solid malignancies.

A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes).

In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.

Ectopic Sox3 activity elicits sensory placode formation.

The induction of sensory organ placodes, in particular the lens placode, represents the paradigm for induction. We show that medaka Sox3 is expressed in the neuroectoderm and in the placodes of all sensory organs prior to placode formation and subsequently in placode-derived tissues. Ectopic Sox3 expression leads to ectopic expression of Pax6 and Eya1 in embryonic ectoderm and causes ectopic lens and otic vesicle formation. The descendants of cells ectopically expressing Sox3-mRNA contribute to ectopic lens tissue. This suggests a permissive role for Sox3 in establishing a placodal competence. In addition, ectopic Sox3 expression leads to the dysgenesis of the endogenous sensory organs. Both effects of ectopic Sox3 expression can be separated by ectopic expression of a truncated Sox3 variant depending on its expression level. Our data suggests that Sox3 is a permissive factor for sensory placode formation and plays an important role in sensory organ development.

A green fluorescent protein enhancer trap screen in Drosophila photoreceptor cells.

The Drosophila ommatidia contain two classes of photoreceptor cells (PR's), the outer and the inner PR's. We performed an enhancer trap screen in order to target genes specifically expressed in PR's. Using the UAS/GAL4 method with enhanced green fluorescent protein (eGFP) as a vital marker, we screened 180000 flies. Out of 2730 lines exhibiting new eGFP patterns, we focused on 16 lines expressing eGFP in particular subsets of PR's. In particular, we describe three lines inserted near the spalt major, m-spondin and furrowed genes, whose respective expression patterns resemble those genes. These genes had not been reported to be expressed in the adult eye. These examples clearly show the ability of our screen to target genes expressed in the adult Drosophila eye.

The transcription factors KNIRPS and KNIRPS RELATED control cell migration and branch morphogenesis during Drosophila tracheal development.

Cell migration during embryonic tracheal system development in Drosophila requires DPP and EGF signaling to generate the archetypal branching pattern. We show that two genes encoding the transcription factors KNIRPS and KNIRPS RELATED possess multiple and redundant functions during tracheal development. knirps/knirps related activity is necessary to mediate DPP signaling which is required for tracheal cell migration and formation of the dorsal and ventral branches. Ectopic knirps or knirps related expression in lateral tracheal cells respecifies their anteroposterior to a dorsoventral migration behavior, similar to that observed in the case of ectopic DPP expression. In dorsal tracheal cells knirps/knirps related activity represses the transcription factor SPALT; this repression is essential for secondary and terminal branch formation. However, in cells of the dorsal trunk, spalt expression is required for normal anteroposterior cell migration and morphogenesis. spalt expression is maintained by the EGF receptor pathway and, hence, some of the opposing activities of the EGF and DPP signaling pathways are mediated by spalt and knirps/knirps related. Furthermore, we provide evidence that the border between cells acquiring dorsal branch and dorsal trunk identity is established by the direct interaction of KNIRPS with a spalt cis-regulatory element.

A transcription unit at the ken and barbie gene locus encodes a novel Drosophila zinc finger protein.

We describe a novel Drosophila transcription unit, located in chromosome region 60A. It encodes a zinc finger protein that is expressed in distinct spatial and temporal patterns during embryogenesis. Its initial expression occurs in a stripe at the anterior and the posterior trunk boundary, respectively. The two stripes are activated and spatially controlled by gap-gene activities. The P-element of the enhancer trap line l(2)02970 is inserted in the 5'-region of the transcript and causes a ken and barbie (ken) phenotype, associated with malformation of male genital structures.

Regulation of Drosophila spalt gene expression.

The region-specific homeotic gene spalt is involved in the specification of terminal versus trunk structures during early Drosophila embryogenesis. Later in development spalt activity participates in specific processes during organogenesis and larval imaginal disc development. The multiple functions of spalt are reflected in distinct spatio-temporal expression patterns throughout development. Here we show that spalt cis-regulatory sequences for region-specific and organ-specific expression are clustered. Their organization may provide the structural basis for the diversification of expression pattern within the spalt/spalt related/spalt adjacent gene complex. We also examined the transacting factor requirement for the blastodermal spalt expression domains. They are under the genetic control of maternal and gap gene products and we show that these products are able to bind to corresponding spalt cis-acting sequences in vitro. The results suggest that the transacting factors, as defined by genetic studies, functionally interact with the spalt regulatory region. In addition, we provide evidence that a zygotic gene product of the terminal system, Tailless, cooperates with the maternal gene product Caudal and thereby activates gene expression in the terminal region of the embryo.

Isolation, characterization, and organ-specific expression of two novel human zinc finger genes related to the Drosophila gene spalt.

The region-specific homeotic gene spalt (sal) of Drosophila specifies head and tail as opposed to trunk segments. During later stages of ontogenesis, sal is also expressed and required in a small number of tissues and organs in the developing embryo. sal encodes a zinc finger protein of unusual but characteristic structure. We made use of these unique features to isolate sal-like genes from humans. Here we report the isolation and molecular characterization of two sal-like transcription units, termed Hsal1 and Hsal2, which are located on chromosomes 16q12.1 and 14q11.1-q12.1, respectively. Their transcripts are expressed in a limited number of adult organs, including the brain. While Hsal2 is evenly expressed in different brain areas, Hsal1 transcripts preferentially accumulate in the corpus callosum and the substantia nigra. In the fetal brain, transcripts of both genes were detected in neurons. The arrangement of sal-like zinc finger domains and their high degree of sequence similarity suggest a novel and conserved subfamily of human zinc finger transcription factors that is closely related to the Drosophila gene product encoded by the gene sal.

Dual function of the region-specific homeotic gene spalt during Drosophila tracheal system development.

We report that the region-specific homeotic gene spalt affects the Drosophila tracheal system at two different stages of embryonic development. Both lack-of-function and gain-of-function experiments show that blastodermal spalt activity restricts tracheal development to 10 bilaterally positioned pairs of tracheal placodes in the trunk region by repressing placode formation in parasegments 2, 3 and 14. The results suggest that the activity of the zinc-finger type transcription factor encoded by spalt suppresses the molecular pathway that establishes tracheal development. spalt function is also necessary for the directed migration of the dorsal trunk cells, a distinct subset of tracheal cells. This process is a prerequisite for the formation of the dorsal trunk generated by fusion of adjacent tracheal metameres into a common tubular structure. The directed cell migration, in which spalt gene function participates, seems to be independent of branch fusion and general tracheal cell migration processes.

Regulation, function and potential origin of the Drosophila gene spalt adjacent, which encodes a secreted protein expressed in the early embryo.

During early embryogenesis of Drosophila the spatial and temporal expression patterns of the region-specific homeotic gene spalt (sal) and the neighbouring gene spalt adjacent (sala) extensively overlap. We show that the initial expression patterns of the two genes in the blastoderm also have identical genetic controls. However, while sal encodes a transcription factor, sala encodes a precursor protein from which a functional signal peptide is cleaved off to generate the secreted sala protein. Ectopic expression or absence of sala protein does not affect embryonic development, adult viability or fertility. In addition to sal and sala, we identified a third gene nearby, termed spalt related (salr), which shares coding sequence similarity and a late embryonic expression pattern with sal, but lacks the early expression domains that are shared by sal and sala. These results suggest that the three genes and their present cis-regulatory regions arose through a chromosomal rearrangement involving local duplication and transposition events in the 32F/33A region on the left arm of the second chromosome.

spalt encodes an evolutionarily conserved zinc finger protein of novel structure which provides homeotic gene function in the head and tail region of the Drosophila embryo.

The region specific homeotic gene spalt (sal) of Drosophila melanogaster promotes the specification of terminal pattern elements as opposed to segments in the trunk. Our results show that the previously reported sal transcription unit was misidentified. Based on P-element mediated germ line transformation and DNA sequence analysis of sal mutant alleles, we identified the transcription unit that carries sal function. sal is located close to the misidentified transcription unit, and it is expressed in similar temporal and spatial patterns during embryogenesis. The sal gene encodes a zinc finger protein of novel structure composed of three widely spaced 'double zinc finger' motifs of internally conserved sequences and a single zinc finger motif of different sequence. Antibodies produced against the sal protein show that sal is first expressed at the blastoderm stage and later in restricted areas of the embryonic nervous system as well as in the developing trachea. The antibodies detect sal homologous proteins in corresponding spatial and temporal patterns in the embryos of related insect species. Sequence analysis of the sal gene of Drosophila virilis, a species which is phylogenetically separated by approximately 60 million years, suggests that the sal function is conserved during evolution, consistent with its proposed role in head formation during arthropod evolution.

Tumoricidal cells increased by pulsating magnetic field.

Repeated applications of pulsed magnetic fields (right-angle waves, 50 Hz = 135 Gauss, 2 Hz = 262 Gauss) significantly enhanced the number and the tumoricidal activity of nonparenchymal liver cells. The transplantable mouse leukemia L1210 used as a tumor model was not significantly influenced, either directly or during Cyclophosphamide treatment.

Tumor cells adhere to intraperitoneal injection sites.

Tumor cells injected intraperitoneally form cell plaques at injection sites in the abdominal wall of mice within a few minutes. Tumor cells appear to be transported passively and chemotactic factors are not involved. Dihydrocortisol blocks cell adherence and silica particles , assumed to destroy macrophages, abolish cell plaque formation.

Studies of erythrocyte sequestration. I. Spleen homing of heat altered erythrocytes.

Heat altered erythrocytes are taken up by spleen macrophages without any signs of mechanical strain as revealed by light and scanning electron microscopy. There was no scintigraphic evidence for changed flow rates within the spleen after sequestration of erythrocytes.

Studies of erythrocyte sequestration. II. Liver homing.

Xenogeneic and enzyme-treated erythrocytes rapidly home to the rat liver as measured by scintigraphic procedures. Histology and scanning electron microscopy revealed that pronase digested erythrocytes are taken up as whole cells by liver histiocytes.