Rodlet cells in Murray cod, Maccullochella peelii peelii (Mitchell), affected with chronic ulcerative dermatopathy.

We have previously identified an unknown cell type in the gills of Murray cod affected with chronic ulcerative dermatopathy (CUD), a condition that causes severe erosion of epidermis surrounding cephalic and lateral line sensory canals. The condition arises in aquaculture facilities that utilize groundwater, with the cause of the condition suggested to be an unknown contaminant(s). Light and transmission electron microscopy were used to characterize and quantify the unknown cells in CUD-affected Murray cod. The cells were identified as rodlet cells and were characterized by their oval or round shape, basally located nucleus, thick fibrillar capsule surrounding the cell, and multiple rodlet sacs containing a central electron-dense core within the cell. Rodlet cells were present in the gills, kidney and intestine of non-CUD-affected and CUD-affected Murray cod; however, differences in the numbers were observed between the groups of fish. A significantly greater number of rodlet cells were observed in the gills and collecting ducts of CUD-affected fish. This is the first report of rodlet cells in Murray cod, and we suggest that the increased rodlet cell numbers in CUD-affected Murray cod may be in response to unknown water contaminant(s) present in the groundwater that give rise to CUD.

Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation.

Methylation of DNA at the dinucleotide CpG is essential for mammalian development and is correlated with stable transcriptional silencing. This transcriptional silencing has recently been linked at a molecular level to histone deacetylation through the demonstration of a physical association between histone deacetylases and the methyl CpG-binding protein MeCP2 (refs 4,5). We previously purified a histone deacetylase complex from Xenopus laevis egg extracts that consists of six subunits, including an Rpd3-like deacetylase, the RbA p48/p46 histone-binding protein and the nucleosome-stimulated ATPase Mi-2 (ref. 6). Similar species were subsequently isolated from human cell lines, implying functional conservation across evolution. This complex represents the most abundant form of deacetylase in amphibian eggs and cultured mammalian cells. Here we identify the remaining three subunits of this enzyme complex. One of them binds specifically to methylated DNA in vitro and molecular cloning reveals a similarity to a known methyl CpG-binding protein. Our data substantiate the mechanistic link between DNA methylation, histone deacetylation and transcriptional silencing.

Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.

CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters.

Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.

Biennial reproductive cycle in an extensive matrotrophic viviparous batoid: the sandyback stingaree Urolophus bucculentus from south-eastern Australia.

Urolophus bucculentus, the largest urolophid species found in southern Australia, exhibits a biennial reproductive cycle. Ovulation occurs during October to January followed by a 15-19 month period of gestation followed by parturition during April to May and a short rest period while the ovarian follicles continue to develop for subsequent ovulation. Male breeding condition peaks during April to June to coincide with the period of parturition. Urolophus bucculentus has the highest matrotrophic contribution reported for any urolophid species, with a mean wet mass gain from egg in utero (4 g) to full-term embryo in utero (250 g) of c. 6250% (maximum c. 7200%), and perhaps explains the biennial female reproductive cycle where 50% of females contribute to each year's recruitment. Litter size (one to five) increases with total length (L(T) ). Females reach a longer maximum L(T) (L(Tmax) ) than do males (885 v. 660 mm). The L(T) at maturity for males and females at 50% mature (L(T50) ) is c. 414 mm (63% of L(Tmax) ) for males and c. 502 mm (57% of L(Tmax) ) for females, length at maternity indicates that recruitment production occurs later in life at c. 632 mm L(T) (71% of L(Tmax) ).

Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alpha v beta 3 integrin to promote epidermal growth factor receptor phosphorylation and growth.

Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matrix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta 3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with alpha v beta 3 integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta 3 integrin ligands in type I collagen. In turn, alpha v beta 3 interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.

Therapeutic potential of RhoA/Rho kinase inhibitors in pulmonary hypertension.

A burgeoning body of evidence suggests that RhoA/Rho kinase (ROCK) signalling plays an important role in the pathogenesis of various experimental models of pulmonary hypertension (PH), including chronic hypoxia-, monocrotaline-, bleomycin-, shunt- and vascular endothelial growth factor receptor inhibition plus chronic hypoxia-induced PH. ROCK has been incriminated in pathophysiologic events ranging from mediation of sustained abnormal vasoconstriction to promotion of vascular inflammation and remodelling. In addition, the 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, statins, which inhibit activation of RhoA by preventing post-translational isoprenylation of the protein and its translocation to the plasma membrane ameliorate PH in several different rat models, and may also be effective in PH patients. Also, phosphorylation of RhoA and prevention of its translocation to the plasma membrane are involved in the protective effect of the type 5-PDE inhibitor, sildenafil, against hypoxia- and bleomycin-induced PH. Collectively, these and other observations indicate that independent of the cause of PH, activation of the RhoA/ROCK pathway serves as a point of convergence of various signalling cascades in the pathogenesis of the disease. We propose that ROCK inhibitors and other drugs that inhibit this pathway might be useful in the treatment of various forms of PH.

A multiple subunit Mi-2 histone deacetylase from Xenopus laevis cofractionates with an associated Snf2 superfamily ATPase.

Chromatin structure plays a crucial regulatory role in the control of gene expression. In eukaryotic nuclei, enzymatic complexes can alter this structure by both targeted covalent modification and ATP-dependent chromatin remodeling. Modification of histone amino termini by acetyltransferases and deacetylases correlates with transcriptional activation and repression [1-3], cell growth [4], and tumorigenesis [5]. Chromatin-remodeling enzymes of the Snf2 superfamily use ATP hydrolysis to restructure nucleosomes and chromatin, events which correlate with activation of transcription [6,7]. We purified a multi-subunit complex from Xenopus laevis eggs which contains six putative subunits including the known deacetylase subunits Rpd3 and RbAp48/p46 [8] as well as substoichiometric quantities of the deacetylase-associated protein Sin3 [9-13]. In addition, we identified one of the other components of the complex to be Mi-2, a Snf2 superfamily member previously identified as an autoantigen in the human connective tissue disease dermatomyositis [14,15]. We found that nucleosome-stimulated ATPase activity precisely copurified with both histone deacetylase activity and the deacetylase enzyme complex. This association of a histone deacetylase with a Snf2 superfamily ATPase suggests a functional link between these two disparate classes of chromatin regulators.

Elastase and matrix metalloproteinase inhibitors induce regression, and tenascin-C antisense prevents progression, of vascular disease.

Increased expression of the glycoprotein tenascin-C (TN) is associated with progression of clinical and experimental pulmonary hypertension. In cultured smooth muscle cells (SMCs) TN is induced by matrix metalloproteinases (MMPs) and amplifies the proliferative response to growth factors. Conversely, suppression of TN leads to SMC apoptosis. We now report that hypertrophied rat pulmonary arteries in organ culture, which progressively thicken in association with cell proliferation and matrix accumulation, can be made to regress by inhibiting either serine elastases or MMPs. This effect is associated with reduced TN, suppression of SMC proliferation, and induction of apoptosis. Selective repression of TN by transfecting pulmonary arteries with antisense/ribozyme constructs also induces SMC apoptosis and arrests progressive vascular thickening but fails to induce regression. This failure is related to concomitant expansion of a SMC population, which produces an alternative cell survival alpha(v)beta(3) ligand, osteopontin (OPN), in response to pro-proliferative cues provided by a proteolytic environment. OPN rescues MMP inhibitor-induced SMC apoptosis, and alpha(v)beta(3) blockade induces apoptosis in hypertrophied arteries. Our data suggest that proteinase inhibition is a novel strategy to induce regression of vascular disease because this overcomes the pluripotentiality of SMC-matrix survival interactions and induces coordinated apoptosis and resorption of matrix.

Tumor necrosis factor alpha and interleukin-1beta regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBP-beta and NF-kappaB.

Manganese superoxide dismutase (MnSOD), a tumor necrosis factor (TNF)-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identified a 238-bp region of intron 2 that was responsive to TNF and interleukin-1beta (IL-1). This TNF response element (TNFRE) had the properties of a traditional enhancer element that functioned in an orientation- and position-independent manner. IVGF of the TNFRE revealed TNF- and IL-1-induced factor occupancy of sites that could bind NF-kappaB and C/EBP. The 5' portion of the TNFRE bound C/EBP-beta in vitro and was both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE bound both NF-kappaB and C/EBP but was not necessary for TNF responsiveness with the MnSOD promoter. However, this 3' portion of the TNFRE was required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter.

Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte.

We investigated the protein associations and enzymatic requirements for the Xenopus histone deacetylase catalytic subunit RPD3 to direct transcriptional repression in Xenopus oocytes. Endogenous Xenopus RPD3 is present in nuclear and cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nuclear. We cloned Xenopus RbAp48 and SIN3 and show that expression of RPD3, but not RbAp48 or SIN3, leads to an increase in nuclear and cytoplasmic histone deacetylase activity and transcriptional repression of the TRbetaA promoter. This repression requires deacetylase activity and nuclear import of RPD3 mediated by a carboxy-terminal nuclear localization signal. Exogenous RPD3 is not incorporated into previously described oocyte deacetylase and ATPase complexes but cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus. We show that RPD3 associates with RbAp48 through N- and C-terminal contacts and that RbAp48 also interacts with SIN3. Xenopus RbAp48 selectively binds to the segment of the N-terminal tail immediately proximal to the histone fold domain of histone H4 in vivo. Exogenous RPD3 may be targeted to histones through interaction with endogenous RbAp48 to direct transcriptional repression of the Xenopus TRbetaA promoter in the oocyte nucleus. However, the exogenous RPD3 deacetylase functions to repress transcription in the absence of a requirement for association with SIN3 or other targeted corepressors.

Gene delivery from a DNA controlled-release stent in porcine coronary arteries.

Expandable intra-arterial stents are widely used for treating coronary disease. We hypothesized that local gene delivery could be achieved with the controlled release of DNA from a polymer coating on an expandable stent. Our paper reports the first successful transfection in vivo using a DNA controlled-release stent. Green fluorescent protein (GFP) plasmid DNA within emulsion-coated stents was efficiently expressed in cell cultures (7.9% +/- 0.7% vs. 0.6% +/- 0.2% control, p < 0.001) of rat aortic smooth muscle cells. In a series of pig stent-angioplasty studies, GFP expression was observed in all coronary arteries (normal, nondiseased) in the DNA-treated group, but not in control arteries. GFP plasmid DNA in the arterial wall was confirmed by PCR, and GFP presence in the pig coronaries was confirmed by immunohistochemistry. Thus, DNA-eluting stents are capable of arterial transfection, and could be useful as delivery systems for candidate vectors for gene therapy of cardiovascular diseases.

Prx1 controls vascular smooth muscle cell proliferation and tenascin-C expression and is upregulated with Prx2 in pulmonary vascular disease.

Prx1 and Prx2 are homeobox transcription factors expressed during vasculogenesis. To begin to elucidate how Prx1 and Prx2 are regulated and function in the adult vasculature, in situ hybridization studies were performed. Prx1 and Prx2 mRNAs were not detected in normal adult rat pulmonary arteries; however, both genes were induced with vascular disease, colocalizing to sites of tenascin-C (TN-C) expression. Because catabolism of the extracellular matrix (ECM) is a critical step in the development of vascular disease, we investigated whether changes in vascular smooth muscle cell (SMC)-ECM interactions regulate Prx1 and Prx2. A10 SMCs cultured on native type I collagen showed low levels of Prx1 and Prx2 mRNA expression, whereas cells cultured on denatured collagen showed higher levels of expression of both genes. At a functional level, transfection of SMCs with a Prx1 expression plasmid significantly increased their growth. Because TN-C also promotes SMC growth and its expression is also upregulated by denatured collagen, we tested and thereafter showed that Prx1 expression significantly enhances TN-C gene promoter activity 20-fold. Similar experiments conducted with truncated Prx1 proteins showed that the N-terminal portion and the homeodomain of Prx1 were necessary to induce the bulk of TN-C promoter activity. These findings support the hypothesis that Prx genes are regulated by changes in SMC adhesion and play key morphoregulatory roles during the development and progression of pulmonary vascular disease in adults.

Uptake and metabolism of 111In-labeled monoclonal antibody B6.2 by the rat liver.

When 111In-labeled murine monoclonal antibodies are used in radio-scintigraphic diagnostic procedures, a large fraction of the injected radionuclide is sequestered by the liver. Neither the cells responsible for the uptake nor the mechanism of uptake are known. Little is known about either the site within the liver of antibody metabolism or the form of the products of metabolism. In these studies, the uptake and metabolism of a monoclonal antibody, B6.2 radiolabeled with 111In or 125I [either intact B6.2 or F(ab')2] were determined in rats. One h after injection of either 125I- or 111In-diethylenetriaminepentaacetic acid (111In-DTPA)-labeled B6.2, the predominant liver cell in which the radionuclide was found was the parenchymal cell. At this time, the absolute uptake of 125I in the liver was 0.23 +/- 0.06% (SD) of the injected dose compared to 0.61 +/- 0.06% when the radionuclide was 111In. Removal of the Fc portion of the antibody reduced the absolute liver uptake of 125I to 0.10 +/- 0.01 and the absolute uptake of 111In to 0.16 +/- 0.06. Both radionuclides were still associated predominantly with the parenchymal cell. Using size exclusion high performance liquid chromatography analysis of liver supernatants the metabolism of radiolabeled B6.2 was followed for 24 h. Of the radioactivity recovered, 47.9% of the 125I was precipitable by centrifugation (and presumed bound to cell membranes) while 15.4% was attached to B6.2 found in the cytosol. In contrast, when 111In-DTPA-B6.2 was administered, 16.0% of 111In recovered from the liver was precipitable by centrifugation, and 6.5% was attached to B6.2 found in the cytosol. Sixty % of the 111In was recovered as a low molecular weight (less than 1000) component in the cytosol. This metabolite was not immunoreactive, nor did it comigrate with ferritin, and was resolved into four components by ion exchange high performance liquid chromatography. Of these, only a minor component cochromatographed with an 111In-DTPA standard. These data suggest that the large accretion of radionuclide by the liver is due to uptake of monoclonal antibodies by an Fc receptor-mediated mechanism and the subsequent accumulation of low molecular weight metabolites, presumably 111In-DTPA, attached to one or more amino acids. The reasons for the entrapment of metabolites in the liver are under investigation.

Correlation of vascular permeability and blood flow with monoclonal antibody uptake by human Clouser and renal cell xenografts.

The specific uptake of 125I-A6H antibody by xenografts of the human renal cell carcinoma (RCC) TK177G in the athymic mouse was considerably greater than that seen for other human tumor xenografts and their associated antibodies (e.g., 125I-B6.2 uptake by the human breast carcinoma, Clouser). In addition the A6H-RCC model also demonstrated both greater localization indices and absolute amount of antibody bound than did the B6.2-Clouser model. Several physiological factors were studied to assess whether they might play a role in this greater specific uptake. Vascular volume was determined using the in situ labeling of red blood cells with 99mTc. Vascular permeability was determined by measuring the amount of 125I-labeled bovine serum albumin and 131I-labeled nonspecific IgG1 (anti-horseradish peroxidase) extravasated out of the tumor vasculature during 1 hr. Relative blood flow to the tumor was determined using the 86Rb method. Blood flow and vascular permeability were found to be significantly greater in the RCC tumor xenografts than in Clouser tumors. Differences in vascular permeability were especially dramatic, showing the vasculature of the RCC xenograft was twice as permeable as that of the Clouser tumor. Animals bearing either RCC or Clouser xenografts were injected with a monoclonal antibody to human major histocompatibility complexes (125I-labeled anti-human histocompatibility complex A, B, C). Tumor uptake of 125I-labeled anti-human histocompatibility complex A, B, C was found to be 5 times greater in RCC than Clouser xenografts. These results, therefore, suggest that the differences seen in the physiological factors studied can account for some of the greater specific 125I-A6H uptake by the RCC tumor than 125I-B6.2 uptake by the Clouser xenograft.

Pharmacokinetics of the monoclonal antibody B72.3 and its fragments labeled with either 125I or 111In.

A comparison of the pharmacokinetics of intact B72.3 (a murine monoclonal antibody specific for human breast and colon carcinoma) with F(ab')2 and Fab fragments labeled with 111In and 125I was done in athymic mice bearing target (LS174T) and non-target (HCT-15) tumors. IgG B72.3 labeled with either isotype imaged LS174T. Biodistributions of both labels were similar in all organs except liver. F(ab')2 also imaged the LS174T tumor, while Fab bearing either isotype did not. The blood clearance was Fab greater than F(ab')2 greater than immunoglobulin G B72.3 for both isotopes. 111In-labeled fragments yielded large accumulations in the kidneys which persisted for 2 days. The different patterns of biodistribution for the various forms of B72.3 labeled with the two isotopes suggest that the most desirable combination of fragment and isotope will depend on the intended use.

Cellular F-actin levels as a marker for cellular transformation: relationship to cell division and differentiation.

Transformation is associated with profound structural and quantitative changes in the cytoskeleton. Herein we report studies using F-actin, a major cytoskeletal protein, as a quantitative marker for transformation cells, focusing on separating the effects of the cell cycle, cell differentiation, and transformation. The model system for these studies consisted of three lymphoblastic cell lines, one untransformed line (RPMI) and two transformed lines, one (HL-60) of which can be induced to differentiate and the other (Daudi) which cannot. The relation of F-actin levels to cell cycle was studied by flow cytometry with the use of fluorescein-phalloidin to label F-actin and propidium iodide to label DNA. F-actin levels in transformed Daudi and HL-60 lines were only two-thirds that of the untransformed RPMI cells. Histograms of the distribution of F-actin showed that the transformed lines consisted of two cell populations, one having an F-actin content near that of untransformed cells and the other having much less. Cell cycle analysis showed that F-actin in untransformed cells increased 10-15% as cells entered the S compartment, remaining approximately constant through G2 + M phases of the cell cycle, but in transformed cells the major increase in F-actin occurred during G2 + M phase. Double-label studies with rhodamine-phalloidin for F-actin and KI-67 monoclonal antibody for dividing cells (cells at late G1, S, G2, and M) measured with quantitative fluorescence image analysis showed that the mean F-actin content of dividing cells was twice that of nondividing cells. These results suggested that most of the cell division-related F-actin increase occurred during late G1 phase in untransformed cells. Differentiation of HL-60 cells with dimethyl sulfoxide or retinoic acid normalized the F-actin content of the nondividing cell population, but dimethyl sulfoxide and retinoic acid produced no detectable change in F-actin in the undifferentiable Daudi cells. A tumor promoter (12-O-tetradecanoylphorphol-13-acetate) inhibits differentiation of hematopoietic cells, resulted in a 32% decrease in the mean F-actin content of RPMI cells due to the appearance of a new subpopulation of low F-actin content. The 12-O-tetradecanoylphorbol-13-acetate-induced changes reversed slowly after removal of 12-O-tetradecanolyphorbol-13-acetate but more rapidly in the presence of retinoic acid. These results indicate that F-actin quantification can serve as a marker for cellular transformation and provides a tool for studying the mechanisms of cellular differentiation that may lead to a better understanding of the oncogenic process.

Cellular F-actin levels as a marker for cellular transformation: correlation with bladder cancer risk.

Previous findings in cultured cells that differentiated cells had markedly higher F-actin levels than undifferentiated cells (Cancer Res., 50: 2215-2220, 1990) suggested that quantitative F-actin measurements in urinary cells might provide diagnostic or prognostic information by identifying those individuals with cells tending towards a lower degree of differentiation. The feasibility of such an approach was investigated using a risk stratification schema. Bladder wash samples were obtained from 163 symptomatic patients being evaluated for bladder cancer and 41 asymptomatic controls without hematuria or other symptoms consistent with bladder cancer. F-actin levels were evaluated by flow cytometry using a fluorescent phalloidin probe. The risk of bladder cancer was stratified according to biopsy, either DNA ploidy by flow cytometry or quantitative fluorescence image analysis cytology, previous bladder cancer history, and hematuria. A strong correlation between the presence of cells with abnormally low F-actin content in cells obtained by bladder wash from 38 patients and biopsy-proved bladder transitional cell carcinoma (P less than 0.001) was observed. A strong correlation was also observed between the presence of cells with low F-actin content and risk of bladder cancer assessed by either stratification schema (P less than 0.0001). The correlation was more consistent with the stratification by quantitative fluorescence image analysis cytology because of the 37% false-positive rate of ploidy analysis by flow cytometry among the control patients. Further evidence that low F-actin was correlated with cellular abnormality was obtained from simultaneously labeling cells for F-actin and with M344 antibody, a monoclonal antibody against a low-grade bladder tumor-related antigen. These studies showed that the F-actin content of the M344-positive cells was lower than that of the M344-negative cells. These results suggest that F-actin could be an early and sensitive marker for bladder cancer detection and risk prognostication.

N-CoR-HDAC corepressor complexes: roles in transcriptional regulation by nuclear hormone receptors.

Many nuclear hormone receptors (NHRs) actively repress the expression of their primary response genes through the recruitment of transcriptional corepressor complexes to regulated promoters. N-CoR and the highly related SMRT were originally isolated and characterized by their ability to interact exclusivelywith the unliganded forms of NHRs and confer transcriptional repression. Recently, both the N-CoR and SMRT corepressors have been found to exist in vivo in multiple, distinct macromolecular complexes. While these corepressor complexes differ in overall composition, a general theme is that they contain histone deacetylase enzymatic activity. Several of these complexes contain additional transcriptional corepressor proteins with functional ties to chromatin structure. Together, these data suggest that modulation of chromatin structure plays a central role in N-CoR mediated transcriptional repression from unliganded NHRs.

Epidermal keratinocyte self-renewal is dependent upon dermal integrity.

The epidermis is a major site of self-renewal in which there is constant replacement by cell division in the basal layers of cells lost by desquamation in the superficial layers. Such a tissue is therefore likely to contain stem cells and in this study we have examined the role of the dermis in the maintenance of epidermal self-renewal. We have developed a mouse model to address the question of whether the maintenance of epidermal self-renewal is dependent, as in the hemopoietic system, upon a heterologous cell type. Intact epidermis separated from dermis at the dermo-epidermal junction or epidermis derived from disaggregated epidermal cells, can reconstitute a stratified squamous epithelium when grafted onto the lumbo-dermal fascia of the mouse or onto an experimentally induced granulation tissue bed. However, we have shown that, after grafting, the clonogenic capacity of the keratinocytes declines sharply and the colonies that are produced are incapable of self-renewal in vitro. Although initially hyperplastic, these epidermal grafts assume an atrophic appearance after 40-70 d and this may be related to the loss of self-renewal observed in vitro. With both experimental murine grafts and clinical grafts the failure of keratinocytes to self-renew can be alleviated, partially, by the presence of the dermis in full-thickness or split-thickness grafts, which implies that the dermis has a functional role in epidermal stem cell maintenance. The relevance of these observations to the clinical experience with cultured autologous keratinocyte sheets as wound dressings to patients is discussed.