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Rearrangements of chromosome band 3q26.2 lead to overexpression of the EVI1 gene and are associated with a poor prognosis in myeloid malignancies. EVI1 is also overexpressed in some cases without 3q26 rearrangements. To uncover its prognostic significance in this patient group, however, it may be necessary to distinguish among several known 5'-end variants of its mRNA. According to a recent report, overexpression of the transcript variant EVI1_1d was associated with shortened survival in acute myeloid leukemia (AML), but overexpression of MDS1/EVI1, whose protein product differs structurally and functionally from that of all other known EVI1 5'-end variants, was not. The aim of the present study was to determine, for the first time, the expression and prognostic significance of all known EVI1 5'-end variants in AML. Quantitative RT-PCR was used to measure the expression of EVI1_1a, EVI1_1b, EVI1_1d, EVI1_3L, and MDS1/EVI1 in 266 samples from patients with de novo AML. To correlate expression of the EVI1 5'-end variants with survival parameters, regression analyses were performed. 41/266 patients (15.4%) overexpressed at least one, but more often several or all, EVI1 transcript type(s). High expression of each of the EVI1 mRNA variants, including MDS1/EVI1, was significantly associated with shortened continuous complete remission in the total patient population as well as in the subgroups of patients with intermediate risk or normal cytogenetics. The present study therefore shows that high levels of each of the known EVI1 mRNA 5'-end variants represents an adverse prognostic factor in de novo AML without 3q26 rearrangements. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat., ((c) 2008 Wiley-Liss, Inc.)

The EVI1 gene codes for a zinc finger transcription factor with important roles both in normal development and in leukemogenesis. Transcriptional activation of this gene through chromosome rearrangements or other, yet to be identified mechanisms leads to particularly aggressive forms of human myeloid leukemia. In vitro as well as in animal model systems, EVI1 affected cellular proliferation, differentiation, and apoptosis in cell type specific ways. Retroviral integrations into the EVI1 locus provided cells with increased abilities to engraft, survive, and proliferate in bone marrow transplantation experiments. Experimental overexpression of EVI1 by itself was insufficient to cause leukemia in animal model systems, but it cooperated with other genes in this process. This review summarizes the currently available experimental evidence for the proposed biochemical and biological functions of this important oncogene.

The EVI1 gene plays important roles in development and leukemogenesis. Recently, human EVI1 has been shown to give rise to at least six different mRNA variants with alternative 5'-ends, only some of which are conserved in mice. In order to gain a basic understanding of the regulation and potential biological importance of these alternative transcripts, we confirmed their expression by Northern blot, and, using real time quantitative RT-PCR, compared their abundance and stability under different conditions. The general expression patterns of the EVI1 5'-end variants in a panel of 20 human tissues were similar, but particularly high or low levels of some of them were noted in certain tissues. Pronounced differences in the expression of the 5'-end variants were noted in response to all-trans retinoic acid: in a human teratocarcinoma cell line, only the EVI1 transcript variants containing alternative exons 1a and 1b were upregulated in response to this agent. This induction required transcriptional activity of RNA polymerase, but was also associated with a substantial increase in the stability of these mRNA variants.

Cystic Fibrosis (CF) is one of the most frequent genetic diseases in the white populations of Europe and North America. Its clinical manifestations are highly variable, ranging from a characteristic life-shortening pathology of the lungs and the pancreas in classical CF to symptoms mainly restricted to male sterility in patients with congenital bilateral aplasia of the vasa deferentia (CBAVD). The genetic basis of CF is mutations in both copies of the CFTR gene, which codes for an ion channel. Even though one single mutation, deltaF508, is responsible for two-thirds of all mutated CFTR alleles, a total of over 1000 CFTR mutations have been described, whose relative frequencies vary between different ethnic groups, and whose biochemical consequences are correlated to the clinical manifestations of the disease. Since 4% of white Europeans and North Americans are healthy heterozygous carriers of CF, the molecular diagnosis of this disease, offered in the context of genetic counselling by a specialist in human genetics, is of great importance.

Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA splicing mechanisms. Using cDNA-based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional exonic splicing enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild-type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue splicing of an enhancer-dependent exon, and found that all seven mutant ESEs had reduced splicing enhancement activity compared to the wild-type sequences. Our results suggest that the wild-type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense and silent mutations, and to understand the structure-function relationship in the corresponding protein., (Copyright 2004 Wiley-Liss, Inc.)

The EVI1 gene in chromosome band 3q26 exhibits a number of properties consistent with a role as an oncogene, and its expression is activated in most myeloid leukemia patients with, as well as in a minority of patients without, 3q26 rearrangements. A splice variant of this gene, MDS1/EVI1, acts as its antagonist at least in some tissue culture assays. We established real-time quantitative reverse transcriptase polymerase chain reaction (RTQ-RT-PCR) assays for these mRNA variants to compare their expression levels in a quantitatively reliable manner. EVI1 was overexpressed to highly variable extents in all patients with, as well as in 14% of patients without, 3q26 rearrangements. In some of these samples, MDS1/EVI1 was also transcribed at elevated levels compared to those of healthy controls. However, although the induction of MDS1/EVI1 was comparable to, or higher than, that of EVI1 in three of five samples with a normal EVI1 locus, this was true for only two of 13 patients with a 3q26 aberration. We further provide preliminary evidence that the RTQ-RT-PCR assay may be useful for disease monitoring in patients overexpressing EVI1., (Copyright 2002 Wiley-Liss, Inc.)

Background and Objectives: Rearrangements of the EVI-1 locus in chromosome band 3q26 are associated with a poor prognosis in myeloid malignancies. To aid the diagnosis of such aberrations, and possibly disease monitoring, we established an interphase fluorescence in situ hybridization (FISH) assay for the affected breakpoint region., Design and Methods: Several overlapping PAC (P1-derived artificial chromosome) clones centromeric to the EVI-1 gene were labeled with a red fluorescent dye, and PAC clones telomeric to EVI-1 with a green fluorochrome. This dual-color probe was hybridized to cytogenetic preparations of cell lines and patients' samples, which were also investigated for the presence of 3q26 rearrangements by chromosome banding analysis., Results: In nuclei without 3q26 rearrangements, two pairs of co-localized red and green signals were observed, while separation of one red/green signal pair or splitting of one red or one green signal was found when 3q26 aberrations were present. The threshold value for true positivity, as determined on 20 samples from patients with myeloid malignancies without 3q26 rearrangements, was 10.2% for separation of one red/green signal pair, and 1% and 1.3% for splitting of one red or one green signal, respectively. In 17 samples from patients with a 3q26 aberration, the percentage of aberrant cells was significantly above these threshold levels., Interpretation and Conclusions: We established an interphase FISH assay that efficiently identifies chromosome breakpoints affecting the EVI-1 locus in 3q26, and represents a useful complement to chromosome banding analysis for the detection of such aberrations.

Chromosome rearrangements affecting band 3q21, namely, the inv(3)(q21q26), the t(3;3)(q21;q26), and the t(1;3)(p36;q21), are associated with a particularly poor prognosis in myeloid leukemia or myelodysplasia. Originally, inv(3) and t(3;3) breakpoints have been reported to cluster in a region (breakpoint cluster region, BCR) of approximately 30 kb, which is located centromeric and downstream of the ribophorin I (RPN-I) gene. More recently, we established a PAC contig that includes the 3q21 BCR, and used these PAC clones to map breakpoints in patient samples by both metaphase and interphase fluorescence in situ hybridization (FISH) analysis. A significant proportion of inv(3) and t(3;3) breakpoints was located at sometimes considerable distances centromeric of the originally described BCR, in a region recently also implicated in t(1;3) rearrangements. These breakpoints may thus define a second, centromeric BCR (BCR-C), or extend the original 3q21 BCR to a size of approximately 100 kb. Activation of the EVI-1 gene in 3q26 by regulatory sequences of the housekeeping gene RPN-I has been suggested as a leukemogenic mechanism in patients with inv(3) and t(3;3). However, despite a number of characteristics that make EVI-1 an attractive candidate oncogene, its biological properties fail to fully explain the phenotype of leukemias carrying 3q rearrangements. Several additional candidate genes have been identified in or near the 3q21 breakpoint region, but their possible contribution to the characteristics of leukemias with 3q21 rearrangements remains to be explored.

In myeloid malignancies, chromosome rearrangements involving band 3q21 are associated with a particularly poor prognosis of the disease. Their sensitive and unequivocal detection is therefore of great clinical importance. In this report, we describe the establishment of an interphase fluorescence in situ hybridization (FISH) assay that complements classical cytogenetic analysis in the diagnosis of such aberrations. PACs that map centromeric and telomeric of known 3q21 breakpoints were labeled with different fluorescent dyes, and the separation of the normally colocalizing signals was used as an indicator of the presence of a 3q21 rearrangement. Two cell lines and 10 primary samples from myeloid leukemia and myelodysplastic syndrome (MDS) patients with 3q21 rearrangements were investigated using the newly established method. The rate of false positivity was determined in 27 control samples from patients with various types of myeloid malignancies. In addition to providing a sensitive and rapid test for the detection of 3q21 aberrations, the interphase FISH assay yields preliminary information about the localization of individual breakpoints. Six of the 10 breakpoints in the patient samples map to an only recently described breakpoint cluster region (BCR) 60 kb centromeric of the originally reported 3q21 BCR. These findings may contribute to the understanding of the molecular basis of the clinical features associated with 3q21 rearrangements., (Copyright 2001 Wiley-Liss, Inc.)

EVI-1 and its variant form, MDS1/EVI1, have been reported to act in an antagonistic manner and be differentially regulated in samples from patients with acute myeloid leukaemia and rearrangements of the long arm of chromosome 3. Here, we show that both EVI-1 and MDS1/EVI1 can repress transcription from a reporter construct containing EVI-1 binding sites and interact with histone deacetylase in mammalian cells. This interaction can be recapitulated in vitro and is mediated by a previously characterized transcription repression domain, whose activity is alleviated by the histone deacetylase inhibitor trichostatin A.

There is evidence that protein kinase C delta (PKCdelta) is a tumor suppressor, although its physiological role has not been elucidated so far. Since important anti-proliferative signals are mediated by cell-cell contacts we studied whether PKCdelta is involved in contact-dependent inhibition of growth in human (FH109) and murine (NIH3T3) fibroblasts. Cell-cell contacts were imitated by the addition of glutardialdehyde-fixed cells to sparsely seeded fibroblasts. Downregulation of the PKC isoforms alpha, delta, epsilon, and mu after prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) resulted in a significant release from contact-inhibition in FH109 cells. Bryostatin 1 selectively prevented TPA-induced PKCdelta-downregulation and reversed TPA-induced release from contact-inhibition arguing for a role of PKCdelta in contact-inhibition. In accordance, the PKCdelta specific inhibitor Rottlerin (1 microM) totally abolished contact-inhibition. Interestingly, immunofluorescence revealed a rapid translocation of PKCdelta to the nucleus when cultures reached confluence with a peak in early-mid G1 phase. Nuclear translocation of PKCdelta in response to cell-cell contacts could also be demonstrated after subcellular fractionation by Western blotting and by measuring PKCdelta-activity after immunoprecipitation. Transient transfection of NIH3T3 cells with a dominant negative mutant of PKCdelta induced a transformed phenotype. We conclude that PKCdelta is involved in contact-dependent inhibition of growth.

The Raf kinases play a key role in relaying signals elicited by mitogens or oncogenes. Here, we report that c-raf-1(-/-) embryos are growth retarded and die at midgestation with anomalies in the placenta and in the fetal liver. Although hepatoblast proliferation does not appear to be impaired, c-raf-1(-/-) fetal livers are hypocellular and contain numerous apoptotic cells. Similarly, the poor proliferation of Raf-1(-/-) fibroblasts and hematopoietic cells cultivated in vitro is due to an increase in the apoptotic index of these cultures rather than to a cell cycle defect. Furthermore, Raf-1- deficient fibroblasts are more sensitive than wild- type cells to specific apoptotic stimuli, such as actinomycin D or Fas activation, but not to tumor necrosis factor-alpha. MEK/ERK activation is normal in Raf-1-deficient cells and embryos, and is probably mediated by B-RAF. These results indicate that the essential function of Raf-1 is to counteract apoptosis rather than to promote proliferation, and that effectors distinct from the MEK/ERK cascade must mediate the anti-apoptotic function of Raf-1.

PKCdelta plays a fundamental role in cell cycle control. Consistent with its proposed tumour suppressor function, ras transfection of the human keratinocyte cell line HaCaT results in a loss of PKCdelta expression mediated by TGFalpha (Exp. Cell Res., 219, 299, 1995). To get more insight into the role of PKCdelta in keratinocytes, we investigated the effects of Rottlerin, a specific inhibitor of protein kinase Cdelta, in HaCaT cells. After Rottlerin treatment, HaCaT cells lost their cobble-stone morphology and displayed a spindle-shaped, fibroblastic phenotype. Additionally, the establishment of cell-cell contacts was prevented. This was caused by an internalization of E-cadherin and beta-catenin as assessed by immunofluorescence. A similar phenotype was observed in the presence of a neutralizing anti-E-cadherin antibody. Rottlerin-treated HaCaT cells proliferated like transformed cells in a three-dimensional cell culture system. We therefore conclude that PKCdelta is involved in mediating cell-cell contacts via E-cadherin and hence regulates differentiation in HaCaT cells., (Copyright 2001 Academic Press.)

Chromosome aberrations affecting band 3q21 are associated with a particularly poor prognosis in patients with acute myeloid leukaemia. To facilitate the molecular characterization of such rearrangements, we established a PAC contig covering the relevant genomic region. Using these PACs as probes in fluorescence in situ hybridization (FISH) experiments, we showed that a number of 3q21 breakpoints in patient samples map to a previously defined 'breakpoint cluster region'. Others, however, are located at varying distances centromeric of it. These results have important implications in the search for genes affected by 3q21 rearrangements.

Rearrangements affecting chromosome band 3q21 are observed in a subgroup of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). However, little is known about the molecular consequences of such aberrations. We therefore established a PAC contig in the 3q21 breakpoint region and identified potential protein coding sequences by exon trapping. One of the exons isolated was from the human GATA-2 gene, which we showed to be transcribed from telomere to centromere. The majority of 3q21 breakpoints are located telomeric to the transcribed portion of this gene in a region that in mice appears to be necessary for proper promoter function. Results of GATA-2 expression analyses in leukemic cell lines as well as primary patient samples are compatible with the hypothesis that 3q21 aberrations contribute to leukemogenesis through deregulation of the hematopoietic transcription factor GATA-2., (Copyright 2000 Academic Press.)

Cessation of division is prerequisite for Schwann cell differentiation but regulation of this critical function is poorly understood. Heregulin/forskolin-induced growth of human Schwann cells (HSCs) in vitro was found to be strongly regulated by cell density and thus could model some aspects of negative growth-regulation in vivo. To better understand this phenomenon, the production of an autocrine growth-inhibitor and the role of contact-inhibition were investigated. The possible involvement of two membrane proteins, contactinhibin (CI) and peripheral myelin protein 22 (PMP22) in regulating growth was studied. Thymidine-labeling of HSCs on collagen-coated dishes was inhibited at cell densities less than one tenth of the density at maximal growth-inhibition. Medium from high density cultures did not inhibit the thymidine-labeling of HSCs at low density, a result that argues against the production of a soluble inhibitor. The expression of CI and PMP22 in nerve and HSCs, and the effect of a function-blocking antibody to CI on HSC growth, were determined. CI was detected in fresh nerve by western blotting, and could easily be detected by immunocytochemistry in cultured HSCs by five days and for several weeks thereafter. Twenty-four hour treatment with anti-CI antibody did not increase the thymidine-labeling of HSCs at any density but a significant increase in HSC number was observed in cultures treated with anti-CI for 20 days. This increase was not related to decreased cell death. PMP22, unlike other myelin proteins, was not down-regulated after nerve dissociation and by seven days nearly all HSCs were PMP22 positive. These results provide evidence for a contact-mediated mechanism of growth-regulation in HSCs and suggest that CI is involved in this mechanism., (Copyright 2000 Wiley-Liss, Inc.)

Growth of non-transformed cells in vitro is regulated by density-dependent mechanisms via cell-cell contacts, leading to arrest in late G1-phase at confluency (contact-inhibition of growth). In the present study it is shown that this results from p16INK4-mediated dissociation of the complex cdk4-cyclin D1, which is responsible for the inactivation of the gate keeper of G1-S transition, the retinoblastoma protein pRb. As a consequence of the inactivation of cdk4, downstream the activation of cdk2 and hyperphosphorylation and thus inactivation of pRb was impaired. Direct evidence for the central role of p16INK4 in growth control comes from the observation that a competitive inhibitor of p16INK4 repressed contact inhibition of growth. These findings provide an explanation for the high incidence of mutation or loss of INK4 in human tumours.

Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-beta (TGF-beta), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-beta inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-beta responses, any role of TGF-beta in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-beta promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant-negative mutant (TbetaRIIDeltacyt) of the TGF-beta type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-beta. In a murine model of bone metastases, expression of TbetaRIIDeltacyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant-negative signaling blockade by expression of a constitutively active TGF-beta type I receptor in the breast cancer cells increased tumor production of parathyroid hormone-related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant-negative TbetaRIIDeltacyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-beta in the development of breast cancer metastasis to bone, via the TGF-beta receptor-mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.

An alternative form of the transcription factor EVI1, MDS1-EVI1, which previously had been believed to exist only in the context of leukemic fusion mRNAs, has recently been shown to be expressed also in normal human tissues. Moreover, it acts as an antagonist of EVI1, activating transcription of reporter constructs repressed by EVI1. We cloned the murine homolog of MDS1-EVI1 as well as mMds1 and show localization of mMds1 close to mEvi1 on chromosome 3. Using RT-PCR, we demonstrate widespread expression of both Evi1 forms in the adult mouse, as well as their upregulation during in vitro hematopoietic differentiation. Our data underscore the biological importance of both EVI1 and MDS1-EVI1 and provide the basis for further studies of their function in the mouse model system., (Copyright 1998 Academic Press.)

In the present work we studied mechanisms of growth control in contact-inhibited and serum-deprived human diploid fibroblasts. The observation that the effects on [3H]thymidine incorporation and reduction of retinoblastoma gene product-phosphorylation were additive when contact-inhibition and serum-deprivation were combined led us to the conclusion that the underlying mechanisms might be different. Both contact-inhibition and serum-deprivation led to a strong decrease of cdk4-kinase-activity and cdk2-phosphorylation at Thr 160, while the total amounts of cdk4 and cdk2 remained constant. In contact-inhibited cells, we revealed a strong protein accumulation of the cdk2-inhibitor p27 and a slight, but significant increase of the cdk4-inhibitor p16. In serum-deprived cells, the protein levels in p27 and p16 remained low. In contrast, we detected a rapid decrease of cyclin D1 and cyclin D3 which did not occur in contact-inhibited cells. These results indicate that serum-deprivation and contact-inhibition have different mechanisms although they affect the same pathway cyclin D-cdk4, pRB, cyclin E-cdk2.

We have identified a novel gene, GR6, located within the leukemia breakpoint region of 3q21, that is normally expressed in early fetal development but not in adult peripheral blood. GR6 is activated in the UCSD-AML1 cell line and in a leukemic sample, both of which carry a t(3;3)(q21;q26). In UCSD-AML1, we have also identified fusion transcripts between the ecotropic viral insertion site I (EVI1) gene in 3q26 and GR6 and between EVI1 and Ribophorin I that maps 30 kb telomeric to GR6 in 3q21. All fusions splice the 5' ends of the 3q21 genes into exon 2 of the EVI1 gene, an event that is similar to the normal intergenic splicing of MDS1-EVI1 and to those previously documented in leukemias with t(3;21) and t(3;12), in which acute myelogenous leukemia 1-EVI1 fusions and ETV6-EVI1 fusions, respectively, occur. The Ribophorin I-EVI1 fusion in particular may be a common occurrence in t(3;3).

We studied the subcellular distribution of cdk2 in synchronized, PDGF-stimulated human fibroblasts (FH109). After contact inhibition and serum depletion, more than 95% of FH109 cells were arrested in G0/G1-phase. PDGF-AB led to a 16-fold increase in proliferation compared with untreated cells. Cell cycle progression was studied by flow cytometric analysis, [3H]thymidine incorporation, and phosphorylation of the retinoblastoma gene product, pRB. Using Western blot analysis after subcellular fractionation, we revealed that after PDGF stimulation the phosphorylated (Thr 160), i.e., activated, form of cdk2 (33 kDa) first appeared in the nucleus at late G1-phase and persisted throughout until to the end of S-phase. Since cdk2 was not synthesized de novo, and the amount of inactive cdk2 (35 kDa) remained constant in the nucleus, we suggested a translocation from the cytosol to the nucleus in late G1. Using immunofluorescence techniques, we detected a diffuse staining in quiescent cells. Starting at late G1-phase, cdk2 immunoreactivity was concentrated to the nucleus while immunoreactivity in the cytosol disappeared. We therefore draw the conclusion that cdk2 is translocated from the cytosol into the nucleus in late G1-phase. Since protein levels and activity of cdk7, which is the catalytic subunit of cdk-activating kinase (CAK) phosphorylating cdk2, remained constant throughout the cell cycle, CAK activity might therefore be regulated by the availability of its substrate cdk2.

Proteases are known to play important roles in cell growth control, although the underlying mechanisms are still poorly understood. Here we show that the protease inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal induced cell cycle arrest in platelet-derived growth factor-stimulated human fibroblasts at the G1/S boundary of the cell cycle by inhibiting the proteasome. Inhibition of the proteasome resulted in accumulation of the tumor suppressor p53, which was followed by an increase in the amount of the cyclin-dependent kinase-inhibitor p21. As a consequence, both phosphorylation and activity of the cyclin-dependent kinase 2/cyclin E complex were inhibited. We further observed that the retinoblastoma gene product, pRb, remained in the hypophosphorylated state, thus preventing cells from progression into the S-phase. These studies strongly support the hypothesis that the proteasome is a key regulator in the G1-phase of cell cycle progression.

Ras proteins play a significant role in signal transduction in response to growth factors and in cell transformation. To be active, ras has to be translocated to the cell membrane. Since subcellular distribution has been mainly studied in vector-transformed cells which highly express ras proteins, and it has been difficult to detect ras in cells expressing the protein at physiological levels, we studied subcellular distribution in human and murine fibroblasts. Here we show for the first time that a significant amount of ras is associated with the membrane skeleton and the cytoskeleton.

Contactinhibin was found to be involved in contact-dependent inhibition of growth. The growth inhibitory activity of contactinhibin is mediated by N-linked oligosaccharides with desialylated beta-glycosidically linked, terminal galactose residues. Here we show that in sparse human fibroblasts contactinhibin was expressed in a biologically inactive, highly sialylated form both on the plasma membrane and intracellularly, while in confluent cells plasma membrane localized contactinhibin was present in a biologically active, low sialylated form. Plasma membranes were shown to contain a glycoprotein sialidase which is suggested to be engaged in the activation of contactinhibin in a cell contact-dependent manner.

The TGF-beta type II receptor (T beta R-II) is a transmembrane serine/threonine kinase that, upon ligand binding, recruits and phosphorylates a second transmembrane kinase, T beta R-I, as a requirement for signal transduction. T beta R-I is phosphorylated by T beta R-II in the GS domain, a 30 amino acid region preceding the kinase domain and conserved in type I receptors for other TGF-beta-related factors. The functional role of seven serines and threonines in the T beta R-I GS domain was investigated by mutational analysis. Five of these residues are clustered (TTSGSGSG) in the middle of the GS domain. Mutation of two or more of these residues impairs phosphorylation and signaling activity. Two additional threonines are located near the canonical start of the kinase domain, and their individual mutation to valine strongly inhibits receptor phosphorylation and signaling activity. Replacement of one of these residues, Thr204, with aspartic acid yields a product that has elevated in vitro kinase activity and signals anti-proliferative and transcriptional responses in the absence of ligand and T beta R-II. The identification of constitutively active T beta R-I forms confirms the hypothesis that this kinase acts as a down-stream signaling component in the TGF-beta receptor complex, and its activation by T beta R-II or by mutation is necessary and sufficient for propagation of anti-proliferative and transcriptional responses.

Background: The number of cells within mammalian tissues is maintained by growth-stimulating and growth-inhibiting mechanisms, with inhibitory signals being superimposed over growth stimuli. This is reflected, in the culture of normal adherent cells, by the phenomenon of density-dependent inhibition of growth: cells cease proliferation after becoming a confluent monolayer. We have shown previously that a plasma membrane glycoprotein, contactinhibin, is a major effector of negative growth regulation. Although transformed cells express contactinhibin in a functionally active form, they are not growth-inhibited, suggesting that the defects that lead to their aberrant growth are located 'downstream' of contactinhibin., Results: Here, we provide evidence that a 92 kD plasma membrane protein, which we call CiR, binds specifically to contactinhibin and acts as a receptor mediating the contact-dependent inhibition of growth of cultured human fibroblasts. When polyclonal antibodies against CiR were introduced into cells using liposomes, confluent cells were released from density-dependent growth control. By contrast, cross-linking CiR that is localized to the plasma membrane, using anti-CiR antibodies, led to growth inhibition, suggesting that CiR is a signalling molecule and implicating CiR oligomerization in signal generation. This conclusion is supported by the finding that binding of contactinhibin by CiR is strongly dependent on the local concentration of both molecules and has a sharp threshold. When CiR was isolated by immuno-precipitation under conditions favouring phosphorylation, it was hyperphosphorylated on serine and threonine residues and had reduced contactinhibin-binding capacity; the binding capacity of CiR was restored after treatment with potato acid phosphatase. Fibroblasts transformed with simian virus 40 had reduced CiR expression, higher CiR phosphorylation levels, and a strongly reduced capacity of CiR to bind to contactinhibin. Phosphatase treatment of the CiR isolated from transformed cells only partially restored its contactinhibin-binding capacity., Conclusions: Homeostasis is the net result of a highly balanced network of growth-stimulating and growth-inhibitory signals. We have shown that density-dependent inhibition of growth in vitro is mediated by the interaction of contactinhibin with a 92 kD plasma membrane glycoprotein, CiR, the contactinhibin-binding capacity of which is regulated by phosphorylation.

Adhesion of tumor cells to endothelial cells is a crucial step in the complex sequence of metastasis. In addition to type and local density of adhesion molecules on both cell types, shear forces exerted by the blood flow have been described to be of major importance in governing cell adhesion. Most of the experiments on the molecular basis of tumor-endothelial cell adhesion have been performed as static assays which lack shear forces. We have developed an artificial venule which shares the following in vivo characteristics. A confluent layer of endothelial cells lines the luminal surface of a glass capillary of 1 mm i.d. with pores of 30 nm diameter to allow diffusion of molecules from outside the capillary. Physiological pressure of 16 mbar, flow rate of 2 cm/s, and shear forces of 2 dynes/cm2 are maintained. This device allowed us to show that under dynamic conditions adhesion of B16 mouse melanoma cells to EA.hy926 endothelial cells is mediated most likely by a lectin-like structure on B16 cells and oligosaccharide(s) on endothelial cells. In addition, endothelial activation-independent adhesion was found to be restricted to only a fraction of endothelial cells, as the number of B16 cells that adhered was independent of the number of B16 cells applied.

Transforming growth factor-beta (TGF-beta) signals by contacting two distantly related transmembrane serine/threonine kinases called receptors I and II. The role of these molecules in signalling has now been determined. TGF-beta binds directly to receptor II, which is a constitutively active kinase. Bound TGF-beta is then recognized by receptor I which is recruited into the complex and becomes phosphorylated by receptor II. Phosphorylation allows receptor I to propagate the signal to downstream substrates. This provides a mechanism by which a cytokine can generate the first step of a signalling cascade.

Transforming growth factor beta (TGF-beta) and activin bind to receptor complexes that contain two distantly related transmembrane serine/threonine kinases known as receptor types I and II. The type II receptors determine ligand binding specificity, and each interacts with a distinct repertoire of type I receptors. Here we identify a new type I receptor for activin, ActR-IB, whose kinase domain is nearly identical to that of the recently cloned TGF-beta type I receptor, T beta R-I. ActR-IB has the structural and binding properties of a type I receptor: it binds activin only in the presence of an activin type II receptor and forms a heteromeric noncovalent complex with activin type II receptors. In Mv1Lu lung epithelial cells, ActR-IB and T beta R-I signal a common set of growth-inhibitory and transcriptional responses in association with their corresponding ligands and type II receptors. The transcriptional responses include elevated expression of fibronectin and plasminogen activator inhibitor 1. Although T beta R-I and ActR-IB are nearly identical in their kinase domains (90% amino acid sequence identity), their corresponding type II receptor kinase domains are very different from each other (42% amino acid sequence identity). Therefore, signaling of a specific set of responses by TGF-beta and activin correlates with the presence of similar type I kinases in their complex. Indeed, other TGF-beta and activin type I receptors (TSR-I and ActR-I) whose kinase domains significantly diverge from those of T beta R-I and ActR-IB do not substitute as mediators of these growth-inhibitory and extracellular matrix transcriptional responses. Hence, we conclude that the type I receptor subunits are primary specifiers of signals sent by TGF-beta and activin receptor complexes.

The gap junctional intercellular communication (GJIC) determined by measuring dye coupling with Lucifer yellow, decreased within 3 d from 66% to 28% in monocultures of rat liver parenchymal cells. Coculturing of the parenchymal cells with a nonparenchymal epithelial cell line from rat liver resulted in increased and stabilized intercellular communication (83% after 3 d). The presence of isolated plasma membrane vesicles of the nonparenchymal epithelial cells also stabilized the intercellular communication between the liver parenchymal cells (70% after 3 d). When liver parenchymal cells were cocultured with a rat liver fibroblast cell line the gap junctional communication between the parenchymal cells was not stabilized (43% after 3 d), and isolated plasma membrane vesicles of the fibroblast were also unable to support the GJIC in parenchymal cells (35% after 3 d). It is concluded that plasma membrane constituents of the nonparenchymal epithelial cells were responsible for the stabilization of the GJIC between parenchymal cells. A heterotypic gap junctional communication between parenchymal and nonparenchymal cells was not observed.

The transforming growth factor beta (TGF-beta) type II receptor (T beta R-II) is a transmembrane serine/threonine kinase that contains two inserts in the kinase region and a serine/threonine-rich C-terminal extension. T beta R-II is required for TGF-beta binding to the type I receptor, with which it forms a heteromeric receptor complex, and its kinase activity is required for signaling by this complex. We investigated the role of various cytoplasmic regions in T beta R-II by altering or deleting these regions and determining the signaling activity of the resulting products in cell lines made resistant to TGF-beta by inactivation of the endogenous T beta R-II. TGF-beta binding to receptor I and responsiveness to TGF-beta in these cells can be restored by transfection of wild-type T beta R-II. Using this system, we show that the kinase insert 1 and the C-terminal tail of T beta R-II, in contrast to the corresponding regions in most tyrosine kinase receptors, are not essential to specify ligand-induced responses. Insert 2 is necessary to support the catalytic activity of the receptor kinase, and its deletion yields a receptor that is unable to mediate any of the responses tested. However, substitution of this insert with insert 2 from the activin receptor, ActR-IIB, does not diminish the ability of T beta R-II to elicit these responses. A truncated T beta R-II lacking the cytoplasmic domain still binds TGF-beta, supports ligand binding to receptor I, and forms a complex with this receptor. However, TGF-beta binding to receptor I facilitated by this truncated T beta R-II fails to inhibit cell proliferation, activate extracellular matrix protein production, or activate transcription from a promoter containing TGF-beta-responsive elements. We conclude that the transcriptional and antiproliferative responses to TGF-beta require both components of a heteromeric receptor complex that differs from tyrosine kinase receptors in its mode of signaling.

Transcription of the Saccharomyces cerevisiae CTT1 gene encoding the cytosolic catalase T has been previously shown to be derepressed by nutrient stress. To investigate whether expression of this gene is also affected by other types of stress, the influence of heat shock on CTT1 expression was studied. The results obtained show that expression of the gene is low at 23 degrees C and is induced rapidly at 37 degrees C. By deletion analysis, a promoter element necessary for high level induction by heat shock was located between base pairs -340 and -364 upstream of the translation start codon. This region was demonstrated to be sufficient for heat shock control by placing it upstream of a S. cerevisiae LEU2-lacZ fusion gene. Mutagenesis of the region showed that the response to heat shock is not mediated by a sequence similar to canonical heat shock elements, but by DNA elements also involved in nutrient control of transcription. Catalase T appears to have a function in protecting yeast cells against oxidative damage under stress conditions. Catalase T-containing strains are less sensitive to exposure to 50 degrees C ("lethal heat shock") than isogenic catalase T-deficient mutants, and catalase T-containing strains pretreated by incubation at 37 degrees C are less sensitive to H2O2 than pretreated catalase-deficient mutants.

Transcription of the CTT1 (catalase T) gene of Saccharomyces cerevisiae is controlled by oxygen via heme, by nutrients via cAMP and by heat shock. Nitrogen limitation triggers a rapid, cycloheximide-insensitive derepression of the gene. Residual derepression in a cAMP-nonresponsive mutant with attenuated protein kinase activity (bcy1 tpk1w tpk2 tpk3) demonstrates the existence of an alternative, cAMP-independent nutrient signaling mechanism. Deletion analysis using CTT1-lacZ fusion genes revealed the contribution of multiple control elements to derepression, not all of which respond to the cAMP signal. A positive promoter element responding to negative control by cAMP was inactivated by deletion of a DNA region between base pairs -340 and -364. Upstream fragments including this element confer negative cAMP control to a LEU2-lacZ fusion gene. Northern analysis of CTT1 expression in the presence or absence of heme, in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23 degrees C) and in heat-shocked cells (37 degrees C) shows that CTT1 is only induced to an appreciable extent when at least two of the three factors contributing to its expression (oxidative stress signaled by heme, nutrient starvation (low cAMP) and heat stress) activate the CTT1 promoter.

Previous studies have shown that plasma membrane compounds are involved in the contact-dependent inhibition of growth of human diploid fibroblasts. The purification of the active plasma membrane glycoprotein is described in this report. The glycoprotein has an apparent molecular mass of 60-70 kD and, due to differential sialylation, isoelectric points between pH 5.5. and 6.2. Treatment with sialidase yielded one spot in two-dimensional gel electrophoresis with an isoelectric point of 6.3. After removal of the N-glycosidically linked oligosaccharide chains, the apparent molecular mass is reduced by approximately 22 kD. Treatment was diluted NaOH, which removes the O-glycosidically linked portion of oligosaccharides, resulted in a reduction of the apparent molecular mass by approximately 5 kD. The addition of 50 ng/ml of this glycoprotein-for which the term "contactinhibin" is proposed-in immobilized form to sparsely seeded human fibroblasts resulted in a reversible 70-80% inhibition of growth. The inhibition was not confined to human fibroblasts as other cells were also inhibited, with the exclusion of transformed cells, which are refractory to contactinhibin. The inhibitory activity was abolished by treatment with beta-galactosidase or glycopeptidase F, indicating that the glycan moiety is the biologically active part of the molecule. Confluent cultures treated with antibodies raised against contactinhibin were released from the contact-dependent inhibition of growth. In addition to enhanced saturation density, these cultures exhibited a crisscross growth pattern and the formation of foci. Immunocytochemical studies showed that contactinhibin was associated with vimentin. Furthermore, contactinhibin was found to be not expressed in a species- or organ-specific manner.

The treatment of human diploid fibroblasts with tumor necrosis factor (TNF)-alpha and with lymphotoxin (LT) is associated with induction of interleukin-6 (IL-6) transcripts with TNF-alpha being 10-fold more potent than LT. Here we report on the TNF-alpha/LT-induced signaling mechanisms responsible for the regulation of IL-6 gene expression in these cells. Run-on assays demonstrated that both TNF-alpha and LT increase IL-6 mRNA levels by transcriptional activation of this gene. Stability studies of IL-6 transcripts in fibroblasts showed that TNF-alpha delayed IL-6 mRNA decay but not LT. The induction of IL-6 transcripts by TNF-alpha and LT was not inhibited by the isoquinoline sulfonamide derivative H7. Similarly, depletion of protein kinase C (PKC) by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) did not change the ability of TNF-alpha and LT to induce IL-6 transcripts, demonstrating that stimulation by these agents may not be mediated by activation of PKC. Stimulation of IL-6 transcripts in fibroblasts did also not require new protein synthesis as exposure to the protein synthesis inhibitor cycloheximide (CHX) enhanced accumulation of IL-6 mRNA in the presence or absence of TNF-alpha or LT.

Growth of normal diploid mammalian cells in vitro is strongly regulated by the actual cell density. Cell-cell contacts via specific plasma membrane glycoproteins whose glycan moieties interact with specific receptors has been found to be a main growth regulatory principle. Malignant growth is suggested to result from impaired function of these receptors.

Treatment of confluent cultures of human diploid fibroblasts with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-7) M) resulted in a 70% reduction of the glutathione (GSH) content, compared with untreated controls. The effect, which was dose-dependent, was observed 8 h after the beginning of the treatment could be followed for up to 72 h. On the other hand, GSH reduction was specific for confluent cultures, as the level of glutathione remained unchanged by TPA treatment of sparse cultures. The addition of immobilized plasma membrane proteins to sparsely seeded cells has been shown previously to induce cellular reactions which are characteristic for confluent cultures. It was shown that TPA treatment of sparse cultures grown in the presence of immobilized plasma membrane proteins also resulted in a 70% reduction of glutathione content. These data agree with the postulated involvement of redox reactions in tumor promotion, and point to a central role of cell-cell contacts in the regulation of biochemical events which are critical in tumorigenesis.

Lectins localized in the plasma membranes seem to be of special importance for the intercellular interaction mechanisms. We describe the isolation of mannose-binding proteins by Triton X-100 extraction and affinity chromatography on agarose-bound mannose. The isolation procedure was performed with whole GH3 cells as well as with isolated plasma membranes. For the isolation of plasma membranes of GH3 cells a mechanical pump was used for the disruption. After differential centrifugation an enriched plasma membrane fraction was achieved by discontinuous sucrose gradient centrifugation. The whole fractionation procedure was controlled by total balance sheets for the marker enzymes of the different cell organelles. The plasma membrane fraction was further characterized by gel electrophoresis and electron microscopy. The SDS gel electrophoresis patterns of the proteins, resulting from the Triton X-100 extraction and the affinity chromatography, are nearly identical for whole cells as well as for the enriched plasma membrane fraction. Therefore we presume these mannose-specific proteins to be plasma membrane bound, showing the molecular properties of integral proteins and having a molecular weight of Mr 67 000, 57 000, 47 000.

Adsorbents for high-performance affinity chromatography were prepared by bonding proteins and reactive Procion triazine dyes to 3-isothiocyanatopropyl- and 3-aminopropylsilicas. The materials prepared were used successfully in the separation of hydrophobic plasma membrane proteins of cultured human fibroblasts. The data obtained show that the reaction of 3-isothiocyanatopropyltriethoxysilane (ITCPS) with the surface hydroxyl groups of silica yields a new and convenient route to preparing an "activated carrier" that is capable of coupling with potential affinity ligands containing amino functional groups. The reaction and bonding procedures of 3-isothiocyanatopropyltriethoxysilane and 3-aminopropyltriethoxysilane with silica were optimized with regard to a controlled and reproducible ligand density by adjusting the type of solvent and organic base as reaction catalyst. The ligand content of reactive triazine dyes directly coupled to 3-aminopropylsilica was significantly higher than that of the 6-aminohexyl derivatives coupled to 3-isothiocyanatopropylsilica. The stability of Procion Blue MX-R bonded to 3-aminopropylsilica and 3-isothiocyanatopropylsilica in phosphate-buffered aqueous solution at pH 5.0 and 8.0 was examined.