Pathologic analysis of capsular and incisional denudation and positive margin status in the development of a robot-assisted laparoscopic prostatectomy program.

The aim of this study is to explore the use of pathologically confirmed capsular incision and denudation as a measure of adequacy of extirpation following robot-assisted laparoscopic prostatectomy (RALP). All patients who underwent RALP at the George Washington University Medical Center during the first 2 years of inception of the robotic prostatectomy program were included. All pathologic specimens were reviewed by a single pathologist. One hundred twenty-eight men who underwent RALP during the first 2 years were identified. Sixty-four patients underwent RALP during the first year (group 1) and all pathologic specimens were reviewed retrospectively. Sixty-four patients underwent RALP during the second year (group 2) after revision of our operative technique and all pathologic specimens were reviewed prospectively. Of patients in group 1, 18 (28%) had a positive surgical margin (PSM), and 18 (28%) with negative surgical margins were found to have capsular incision or denudation. In group 1, 32 (50%) patients had evidence of iatrogenic capsular violation. Group 2 consisted of 13 (20%) patients with a PSM and 9 (14%) margin-negative patients with capsular incision or denudation. Group 2 had a total of 22 (34%) patients with evidence of iatrogenic capsular violation. Overall reduction in positive margins was not statistically significant between the groups. Improvement in capsular incision/denudation rate and overall capsular violation between the two groups was statistically significant (P < 0.03 and <0.0055). Surgical margin status alone underestimates the overall quality of surgical resection after RALP because not all capsular violations result in a PSM. Surgeon-guided pathologic review in addition to intraoperative experience may improve oncologic success during the RALP learning curve.

In vivo requirements for GATA-1 functional domains during primitive and definitive erythropoiesis.

GATA-1 is a transcription factor essential for erythroid/megakaryocytic cell differentiation. To investigate the contribution of individual domains of GATA-1 to its activity, transgenic mice expressing either an N-terminus, or an N- or C-terminal zinc finger deletion of GATA-1 (Delta NT, Delta NF or Delta CF, respectively) were generated and crossed to GATA-1 germline mutant (GATA-1.05) mice. Since the GATA-1 gene is located on the X-chromosome, male GATA-1 mutants die by embryonic day 12.5. Both Delta NF and Delta CF transgenes failed to rescue the GATA-1.05/Y pups. However, transgenic mice expressing Delta NT, but not the Delta NF protein, were able to rescue definitive hematopoiesis. In embryos, while neither the Delta CF protein nor a mutant missing both N-terminal domains (Delta NTNF) was able to support primitive erythropoiesis, the two independent Delta NT and Delta NF mutants could support primitive erythropoiesis. Thus, lineage-specific transgenic rescue of the GATA-1 mutant mouse revealed novel properties that are conferred by specific domains of GATA-1 during primitive and definitive erythropoiesis, and demonstrate that the NT and NF moieties lend complementary, but distinguishable properties to the function of GATA-1.

Hand-assisted laparoscopic radical nephrectomy: the experience of the inexperienced.

PURPOSE: We retrospectively examined the experience of novice laparoscopic surgeons performing hand-assisted laparoscopic radical nephrectomy. The purpose was to determine if urologists with minimal laparoscopic training could perform hand-assisted laparoscopic nephrectomies in a safe and efficient manner. MATERIALS AND METHODS: The first six hand-assisted laparoscopic radical nephrectomies performed by four different urology residents at the Chicago Lakeside VA hospital were reviewed. The residents included three chief urology residents and one postgraduate year 3 urology resident. None of the residents had taken any laparoscopic course, and all had limited exposure to the hand-assisted technique. In all cases, the residents performed the entire operation. The patients were evaluated for operative time, tumor size, body mass index, and ASA score. RESULTS: All six procedures were completed without conversion to the open technique. The average operating time was 215.8 minutes, and the time from incision to organ removal averaged 140.8 minutes. The average estimated blood loss was 166 mL. Complications included an intraoperative diaphragmatic injury (recognized and repaired laparoscopically) and one postoperative ileus. CONCLUSION: Hand-assisted laparoscopic radical nephrectomy can be performed safely and efficiently by urologists with minimal laparoscopic experience.

Reconstitution of human beta-globin locus control region hypersensitive sites in the absence of chromatin assembly.

The human beta-globin genes are regulated by the locus control region (LCR), an element composed of multiple DNase I-hypersensitive sites (HS sites) located 5' to the genes. Various functional studies indicate that the LCR confers high-level, position-independent, and copy number-dependent expression to linked globin genes in transgenic mice. However, the structural basis for LCR function is unknown. Here we show that LCR HS sites can be reconstituted in an erythroid cell-specific manner on chromatin-assembled LCR templates in vitro. Surprisingly, HS2 and HS3 are also formed with erythroid proteins in the absence of chromatin assembly, indicating that sensitivity to nucleases is not simply a consequence of nucleosome reorganization. The generation of LCR HS sites in the absence of chromatin assembly leads to the formation of S1- and KMnO(4)-sensitive regions in HS2 and HS3. These sites are also sensitive to S1 nuclease in erythroid cells in vivo, suggesting a distorted DNA structure in the LCR core enhancer elements. Finally, we show that RNA polymerase II initiates transcription in the HS2 and HS3 core enhancer regions in vitro. Transcription in both HS2 and HS3 proceeds in a unidirectional manner. Taken together, the data suggest that erythroid proteins interact with the core enhancer elements, distort the DNA structure, and recruit polymerase II transcription complexes. These results further our understanding of the structural basis for LCR function and provide an explanation for why the LCR core regions are so extremely sensitive to nucleases in erythroid cells.

Context-dependent EKLF responsiveness defines the developmental specificity of the human epsilon-globin gene in erythroid cells of YAC transgenic mice.

We explored the mechanism of definitive-stage epsilon-globin transcriptional inactivity within a human beta-globin YAC expressed in transgenic mice. We focused on the globin CAC and CAAT promoter motifs, as previous laboratory and clinical studies indicated a pivotal role for these elements in globin gene activation. A high-affinity CAC-binding site for the erythroid krüppel-like factor (EKLF) was placed in the epsilon-globin promoter at a position corresponding to that in the adult beta-globin promoter, thereby simultaneously ablating a direct repeat (DR) element. This mutation led to EKLF-independent epsilon-globin transcription during definitive erythropoiesis. A second 4-bp substitution in the epsilon-globin CAAT sequence, which simultaneously disrupts a second DR element, further enhanced ectopic definitive erythroid activation of epsilon-globin transcription, which surprisingly became EKLF dependent. We finally examined factors in nuclear extracts prepared from embryonic or adult erythroid cells that bound these elements in vitro, and we identified a novel DR-binding protein (DRED) whose properties are consistent with those expected for a definitive-stage epsilon-globin repressor. We conclude that the suppression of epsilon-globin transcription during definitive erythropoiesis is mediated by the binding of a repressor that prevents EKLF from activating the epsilon-globin gene.

GATA2 is required for the generation of V2 interneurons.

During embryogenesis, transcription factor GATA2 is expressed in a variety of distinct cell types, and earlier experiments showed that GATA2 is a vital regulator of both hematopoiesis and urogenital development. Despite the fact that GATA2 is expressed early and abundantly in the nervous system, there has been no demonstration of its direct participation in neurogenesis. We show here that GATA2 is expressed in the ventral spinal cord exclusively in newly generated V2 interneurons, suggesting that GATA2 might be required for the generation of this discrete neuronal population. Proof for this hypothesis was provided by showing that the number of cells expressing V2 neuronal markers was drastically diminished in gata2 null mutant embryos. The tissue-specific enhancer that directs gata2 transcription specifically in V2 neurons was localized to a 190 bp intragenic element lying within gata2 intron 5, and this element is both necessary and sufficient to confer GATA2 spinal cord expression. The identification of a V2-specific enhancer should allow fundamental new insight into the genetic hierarchy of regulatory events that govern neurogenesis in a well-defined cell lineage.

GATA factor transgenes under GATA-1 locus control rescue germline GATA-1 mutant deficiencies.

GATA-1 germline mutation in mice results in embryonic lethality due to defective erythroid cell maturation, and thus other hematopoietic GATA factors do not compensate for the loss of GATA-1. To determine whether the obligate presence of GATA-1 in erythroid cells is due to its distinct biochemical properties or spatiotemporal patterning, we attempted to rescue GATA-1 mutant mice with hematopoietic GATA factor complementary DNAs (cDNAs) placed under the transcriptional control of the GATA-1 gene. We found that transgenic expression of a GATA-1 cDNA fully abrogated the GATA-1-deficient phenotype. Surprisingly, GATA-2 and GATA-3 factors expressed from the same regulatory cassette also rescued the embryonic lethal phenotype of the GATA-1 mutation. However, adult mice rescued with the latter transgenes developed anemia, while GATA-1 transgenic mice did not. These results demonstrate that the transcriptional control dictating proper GATA-1 accumulation is the most critical determinant of GATA-1 activity during erythropoiesis. The results also show that there are biochemical distinctions among the hematopoietic GATA proteins and that during adult hematopoiesis the hematopoietic GATA factors are not functionally equivalent.

Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system.

Mouse embryos deficient in Gata3 die by 11 days post coitum (d.p.c.) from pathology of undetermined origin. We recently showed that Gata3-directed lacZ expression of a 625-kb Gata3 YAC transgene in mice mimics endogenous Gata3 expression, except in thymus and the sympathoadrenal system. As this transgene failed to overcome embryonic lethality (unpublished data and ref. 3) in Gata3-/- mice, we hypothesized that a neuroendocrine deficiency in the sympathetic nervous system (SNS) might cause embryonic lethality in these mutants. We find here that null mutation of Gata3 leads to reduced accumulation of Th (encoding tyrosine hydroxylase, Th) and Dbh (dopamine beta-hydroxylase, Dbh) mRNA, whereas several other SNS genes are unaffected. We show that Th and Dbh deficiencies lead to reduced noradrenaline in the SNS, and that noradrenaline deficiency is a proximal cause of death in mutants by feeding catechol intermediates to pregnant dams, thereby partially averting Gata3 mutation-induced lethality. These older, pharmacologically rescued mutants revealed abnormalities that previously could not be detected in untreated mutants. These late embryonic defects include renal hypoplasia and developmental defects in structures derived from cephalic neural crest cells. Thus we have shown that Gata3 has a role in the differentiation of multiple cell lineages during embryogenesis.

One enhancer mediates mafK transcriptional activation in both hematopoietic and cardiac muscle cells.

Members of the small Maf family of transcription factors play important roles in hematopoiesis. Using transgenic assays, we discovered a tissue-specific enhancer 3' to the mafK gene. This enhancer directs mafK transcription in hematopoietic as well as in developing cardiac muscle cells, and was thus designated the hematopoietic and cardiac enhancer of mafK (HCEK). Only two of four GATA consensus motifs identified within HCEK contributed to enhancer activity, and both of these sites were required for both cardiac and hematopoietic transcriptional activation. The expression profile of MafK significantly overlapped that of GATA-1 in hematopoietic cells and of GATA-4/-6 in cardiac tissues. Each of these GATA factors bound with high specificity to both of the critical GATA sites in HCEK. Hence, the mafK gene is regulated by different GATA proteins in the hematopoietic and cardiac compartments through the same two GATA-binding sites in HCEK. These data provide the first in vivo demonstration that distinct members of a related transcription factor family activate the tissue-specific expression of a single target gene using the same cis-regulatory element.

Perinatal synthetic lethality and hematopoietic defects in compound mafG::mafK mutant mice.

Prior studies exploring the mechanisms controlling erythroid gene regulation implicated MARE (Maf recognition element) cis-elements as crucial to the transcriptional activity of many erythroid genes. Numerous transcription factors can elicit responses through MAREs, including not only the AP-1 family proteins, but also a growing list of factors composed of Cap-N-Collar (CNC)-small Maf heterodimers. While these factors can activate transcription from MAREs in co-transfection assays, mouse germline mutations in cnc genes tested to date have failed to reveal primary erythroid phenotypes. Here we report that after combining the mafK and mafG targeted null alleles, mutant animals display several synthetic phenotypes, including erythroid deficiencies. First, compound homozygous small maf gene mutants survive embryogenesis, but die postnatally. Secondly, compound mutant animals develop severe neurological disorders. Thirdly, they exhibit an exacerbated mafG deficiency in megakaryopoiesis, specifically in proplatelet formation, resulting in profound thrombocytopenia. Finally, the compound mutant animals develop severe anemia accompanied by abnormal erythrocyte morphology and membrane protein composition. These data provide direct evidence that the small Maf transcription factors play an important regulatory role in erythropoiesis.

Beta-globin YAC transgenes exhibit uniform expression levels but position effect variegation in mice.

Expression of a construct integrated at different genomic locations often varies because of position effects that have been subcategorized as stable (decreased level of expression) and variegating (decreased proportion of expressing cells). It is well established that locus control regions (LCRs) generally overcome position effects in transgenes. However, whether stable and variegated position effects are equally overcome by an intact LCR has not been determined. We report that single-copy yeast artificial chromosome transgenes containing an unmodified human beta -globin locus were not subject to detectable stable position effects but did undergo mild to severe variegating position effects at three of the four non-centromeric integration sites tested. We also find that, at a given integration site, the distance and the orientation of the LCR relative to the regulated gene contributes to the likelihood of variegating position effects, and can affect the magnitude of its transcriptional enhancement. DNase I hypersensitive site (HSS) formation varies with the proportion of expressing cells, not the level of gene expression, suggesting that silencing of the transgene is associated with a lack of HSS formation in the LCR region. We conclude that transcriptional enhancement and variegating position effects are caused by fundamentally different but inter-dependent mechanisms.

In vivo modulation of human beta-globin gene switching.

Continuing accumulation of information from the genome projects requires parallel development of technologies to assess the in vivo functions of conserved sequences. We can now manipulate huge DNA molecules (such as yeast artificial chromosomes; YACs) in vivo, permitting the analysis of very large single genes or multigene loci as they exist in the chromosome. However, since transgenes integrate randomly into different chromatin environments, accurate evaluation of gene function in such transgenic mice is still fraught with pitfalls. We recently developed the use of cre-mediated homologous recombination to manipulate YAC transgenic mice in vivo, and successfully applied this strategy to the analysis of human beta-globin locus regulation. Here we describe these results and discuss the application of this technology to the analysis of this and related problems.

Positive or negative MARE-dependent transcriptional regulation is determined by the abundance of small Maf proteins.

The small Maf transcription factor proteins bind to Maf Recognition Elements (MAREs) by dimerizing with CNC proteins or themselves. We undertook experiments to clarify the functional relationship between the small Mafs and their partners in vivo. Embryos expressing abundant transgene-derived MafK died of severe anemia, while lines expressing lower levels of small Maf lived to adulthood. Megakaryocytes from the latter overexpressing lines exhibited reduced proplatelet formation and MARE-dependent transcription, phenocopying mafG null mutant mice. When the mafG null mutants were bred to small Maf-overexpressing transgenic animals, both loss- and gain-of-function phenotypes were reversed. These results provide direct in vivo evidence that transcriptional regulation through MARE elements hinges on an exquisitely sensitive balance of activating CNC molecules and their small Maf partners.

The transcription factor GATA3 is a downstream effector of Hoxb1 specification in rhombomere 4.

In this paper, we show that the transcription factor GATA3 is dynamically expressed during hindbrain development. Function of GATA3 in ventral rhombomere (r) 4 is dependent on functional GATA2, which in turn is under the control of Hoxb1. In particular, the absence of Hoxb1 results in the loss of GATA2 expression in r4 and the absence of GATA2 results in the loss of GATA3 expression. The lack of GATA3 expression in r4 inhibits the projection of contralateral vestibuloacoustic efferent neurons and the migration of facial branchiomotor neurons similar to Hoxb1-deficient mice. Ubiquitous expression of Hoxb1 in the hindbrain induces ectopic expression of GATA2 and GATA3 in ventral r2 and r3. These findings demonstrate that GATA2 and GATA3 lie downstream of Hoxb1 and provide the first example of Hox pathway transcription factors within a defined population of vertebrate motor neurons.

The polyoma virus enhancer cannot substitute for DNase I core hypersensitive sites 2-4 in the human beta-globin LCR.

The polyoma virus enhancer (PyE) is capable of conferring integration position-independent expression to linked genes in stably transfected erythroid cells after joining to DNase I hypersensitive site (HS) 5 of the human beta-globin locus control region (LCR). In attempting to separate the chromatin opening activity of the LCR from its enhancer activity and to investigate contributions of the individual HS core elements to LCR function, the human beta-globin LCR HS2, HS3 and HS4 core elements were replaced with the PyE within the context of a yeast artificial chromosome (YAC) bearing the whole locus. We show here that, in contrast to its function in cultured cells, the PyE is unable to replace HS core element function in vivo. We found that the PyE substitution mutant LCR is unable to provide either chromatin opening or transcriptional potentiating activity at any erythroid developmental stage in transgenic mice. These data provide direct evidence that the human beta-globin LCR core elements specify unique functions that cannot be replaced by a ubiquitous enhancer activity.

Transforming growth factor-beta type II receptor confers tumor suppressor activity in murine renal carcinoma (Renca) cells.

OBJECTIVES: To demonstrate that the introduction of the transforming growth factor-beta (TGF-beta) type II receptor (TbetaR-II) decreases tumorigenicity in an aggressive murine renal carcinoma line, Renca. These cells do not express TbetaR-II. Because the presence of TbetaR-II in benign epithelial cells is ubiquitous, the ability to restore tumor suppressor activity in the Renca cell line with its introduction would elucidate the role of TbetaR-II as a tumor suppressor gene. METHODS: Renca cells were stably transfected with a retrovirus-mediated TbetaR-II expression vector. In vitro sensitivity to growth inhibitory effect of TGF-beta was assessed by the 3H-thymidine incorporation assay. For in vivo testing, xenograft tumors were produced by subcutaneous injection of tumor cells into immunodeficient nude mice. The tumorigenicity of these TbetaR-II transfected cells was tested. Wild-type Renca cells and cells transfected with the control vector were also tested for comparison. RESULTS: Expression of TbetaR-II mRNA was evident in Renca cells after transfection with the TbetaR-II construct. In vitro sensitivity to the growth inhibitory effect of TGF-beta was restored. This effect of TGF-beta was reversible with a neutralizing antibody specific for the extracellular domain of TbetaR-II. Xenografts grown from TbetaR-II transfected cells were significantly smaller, weighed less, and developed tumors later than those developed from wild-type Renca cells and those transfected with the control vector. CONCLUSIONS: We conclude that TbetaR-II is a central mediator of tumorigenicity in Renca cells. As with other tumor suppressor genes, the loss of TbetaR-II expression allows for the development of an aggressive phenotype.

Characterization of the murine mafF gene.

Small Maf proteins are obligatory heterodimeric partner molecules of mammalian Cap'n'Collar proteins that together control a wide variety of eukaryotic genes. Although both MafK and MafG are expressed in overlapping but distinct tissue distribution patterns during embryonic development, the physiological consequences of loss-of-function mutations in either gene are modest. This suggested that compensation by the third small Maf protein, MafF, might be a major reason for such mild phenotypes and that further analysis of MafF might therefore provide important insights for understanding small Maf regulatory function(s). We therefore cloned, mapped, transcriptionally and developmentally characterized, and finally disrupted the mafF gene. We show that murine mafF is transcriptionally regulated by three different promoters and is most abundantly expressed in the lung. The lacZ gene inserted into the mafF locus revealed prominent expression sites in the gut, lung, liver, outflow tract of the heart, cartilage, bone membrane, and skin but not in hematopoietic cells at any developmental stage. Homozygous mafF null mutant mice were born in a normal Mendelian ratio and displayed no obvious functional deficiencies, indicating that MafF activity may be dispensable even in tissues where the expression of other small Maf proteins is quite low.

The mouse GATA-2 gene is expressed in the para-aortic splanchnopleura and aorta-gonads and mesonephros region.

We previously reported that the mouse GATA-2 gene is regulated by two alternative promoters (Minegishi et al, J Biol Chem, 273:3625, 1998). Although the more proximal IG (general) promoter is active in almost all GATA-2-expressing cells, the distal IS (specific) promoter activity was selectively detected in hematopoietic tissues but not in other mesodermal tissues. We report here in vivo analysis of the GATA-2 locus and its regulatory characteristics in hematopoietic tissues of transgenic mice. Transgenes containing 6 or 7 kbp of sequence flanking the 5' end of the IS first exon direct expression of beta-galactosidase or green fluorescent protein (GFP) reporter genes specifically to the para-aortic splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the neural tissues. In situ hybridization analysis showed that reporter gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45 antigens were identified in the GFP-positive cells from the AGM region and fetal liver, indicating that GATA-2 is expressed in immature hematopoietic cells. Deletion of 3.5 kbp from the 5' end of the 6.0 kbp IS promoter construct, including one of the DNase I hypersensitive sites, completely abolished hematopoietic expression. These experiments describe an early developmental GATA-2 hematopoietic enhancer located between 6.0 and 2.5 kbp 5' to the IS exon.