Biomedical subjects

D V Faller

Publications and source records attributed to D V Faller.

At least 37 records · Page 2Linked to original sources

PubMed1999

Establishment of order in the flow of genetic information in cells.

The activities related to the flow of genetic information encoded in DNA in a cell are very orderly. This order, in a living cell, is achieved through specific, but noncovalent, interactions of varieties of structurally dynamic macromolecules under constantly changing physiological conditions. Hence, it is expected that there should be some force that can stabilize the multicomponent reaction processes and establish (or maintain) order in genetic regulatory functions under far-from-equilibrium conditions. The genetic regulatory functions in a cell, however, are believed to be energetically coupled. Expression of genes in a cell is often modulated under changing environmental conditions, raising the possibility of a state controlled nature of the genetic regulatory functions. Adenosine triphosphate (ATP) is the major free-energy contributor for these energy-consuming cellular activities. Enzymatic transfer of high-energy phosphate group from ATP to other reactive components is considered to be the chief mode of energy-transduction in a cell for various biosynthetic processes, as well as other activities related to the flow of information. In an effort to find a solution of the paradox, we assessed the contribution of physiological state of a cell in the process of maintaining order in genetic regulatory functions. As an approach, we systematically perturbed the normal energy flow of a cellular system (bovine aortic endothelial [BAE] cell) by a protein kinase inhibitor (staurosporine), and then followed the expression patterns of several constitutively-expressed protein-encoding genes to measure the effects. Staurosporine, as a function of its concentration, disintegrated the membrane structure of these cells, and eventually caused their death. These secondary consequences of staurosporine treatment offered two additional grossly altered physiological states of the cell to study. Under all of these dramatically altered energy states of the system, an extreme degree of functional coherence prevailed at every level of genetic regulatory function. Integrity at the level of gene transcription remained unaffected. Degradation rate of specific mRNA remained unaltered. Translational activities involving varieties of mRNA species continued in an well-ordered manner. Other state changes, resulting from nutrient and metabolic starvation, or inhibition of oxidative phosphorylation, in addition to the staurosporine treatments, also failed to disintegrate these ordered activities. The steady-state levels of specific mRNA underwent certain changes in these conditions, however, without maintaining any proportional relationships with the staurosporine concentrations applied or the ATP levels in the cell. These results thus led us to propose that the internal energy or a certain intrinsic property of the participating components, rather than the physiological state of the cell, acts as the dominant force in maintaining order and stability of genetic regulatory functions in a cell. Kinetic analyses under different energy states of the cell also supported the hypothesis, and further demonstrated the autoregulatory nature of the genetic order establishment. All of these results suggest a process of molecular self-organization as the fundamental principle for genetic regulation in a cellular system.

Actins↗

PubMed1999

Endothelial cell responses to hypoxic stress.

1. Changes in the environmental oxygen tension to which cells are exposed in vivo result in physiological and sometimes pathological consequences that are associated with differential expression of specific genes. 2. Low oxygen tension (hypoxia) affects endothelial cellular physiology in vivo and in vitro in a number of ways, including the transcriptionally regulated expression of vasoactive substances and matrix proteins involved in modulating vascular tone or remodelling the vasculature and surrounding tissue. 3. Hypoxia results in the transcriptional induction of genes encoding vasoconstrictors and smooth muscle mitogens (PDGF-B, endothelin-1, VEGF, thrombospondin-1) and genes encoding matrix or remodelling molecules (collagenase IV (MMP-9), thrombospondin-1) and reciprocal transcriptional inhibition of vasodilatory or anti-mitogenic effectors (eNOS). 4. Oxygen appears to signal through a novel haem-containing sensor and signals initiated by this sensor alter the levels and DNA-binding activity of transcription factors such as activating protein (AP)-1, nuclear factor-kappa B and hypoxia-inducible transcription factor-1. 5. The genes encoding vasoactive factors regulated by oxygen tension are themselves also regulated by the vasoactive agent nitric oxide (NO). 6. Nitric oxide and oxygen transduce similar signals (i.e. their absence results in identical patterns of gene expression in endothelial cells and other cell types). 7. Thus, NO can feedback on and modulate signals induced by hypoxia and vice versa. For example, NO, which can act directly on smooth muscle cells as a vasodilator, can also facilitate vasodilation indirectly by reversing the production of vasoconstrictors induced by hypoxia. 8. Short-term exposure of endothelial cells to low oxygen tension results in the elaboration of predominantly vasoconstricting effectors, while longer-term and more severe hypoxic exposure generates factors that can induce smooth muscle proliferation and remodelling. 9. Thus, the endothelial cell response to hypoxic stress can result in two different consequences in the surrounding tissues, depending on the duration of the exposure: short-term exposure causes physiological and reversible modulation of vascular tone and blood flow; chronic hypoxic stress results in irreversible remodelling of the vasculature and surrounding tissues, with smooth muscle proliferation and fibrosis. 10. This dichotomy of responses to hypoxia may explain, in part, both the acute and chronic pathophysiological sequelae of diseases characterized by regional hypoxia, including atherosclerosis, pulmonary hypertension, sickle cell disease and systemic sclerosis (scleroderma).

Cell Hypoxia↗

PubMed1999

Feline leukemia virus long terminal repeat activates collagenase IV gene expression through AP-1.

Leukemia and lymphoma induced by feline leukemia viruses (FeLVs) are the commonest forms of illness in domestic cats. These viruses do not contain oncogenes, and the source of their pathogenic activity is not clearly understood. Mechanisms involving proto-oncogene activation subsequent to proviral integration and/or development of recombinant viruses with enhanced replication properties are thought to play an important role in their disease pathogenesis. In addition, the long terminal repeat (LTR) regions of these viruses have been shown to be important determinants for pathogenicity and tissue specificity, by virtue of their ability to interact with various transcription factors. Previously, we have shown that, in the case of Moloney murine leukemia virus, the U3 region of the LTR independently induces transcriptional activation of specific cellular genes through an LTR-generated RNA transcript (S. Y. Choi and D. V. Faller, J. Biol. Chem. 269:19691-19694, 1994; S.-Y. Choi and D. V. Faller, J. Virol. 69:7054-7060, 1995). In this report, we show that the U3 region of exogenous FeLV LTRs can induce transcription from collagenase IV (matrix metalloproteinase 9) and monocyte chemotactic protein 1 (MCP-1) promoters up to 12-fold. We also show that AP-1 DNA-binding activity and transcriptional activity are strongly induced in cells expressing FeLV LTRs and that LTR-specific RNA transcripts are generated in those cells. Activation of mitogen-activated protein kinase kinases 1 and 2 (MEK1 and -2) by the LTR is an intermediate step in the FeLV LTR-mediated induction of AP-1 activity. These findings thus suggest that the LTRs of FeLVs can independently activate transcription of specific cellular genes. This LTR-mediated cellular gene transactivation may play an important role in tumorigenesis or preleukemic states and may be a generalizable activity of leukemia-inducing retroviruses.

3T3 Cells↗

PubMed1998

Alterations in protein-DNA interactions in the gamma-globin gene promoter in response to butyrate therapy.

The mechanisms by which pharmacologic agents stimulate gamma-globin gene expression in beta-globin disorders has not been fully established at the molecular level. In studies described here, nucleated erythroblasts were isolated from patients with beta-globin disorders before and with butyrate therapy, and globin biosynthesis, mRNA, and protein-DNA interactions were examined. Expression of gamma-globin mRNA increased twofold to sixfold above baseline with butyrate therapy in 7 of 8 patients studied. A 15% to 50% increase in gamma-globin protein synthetic levels above baseline gamma globin ratios and a relative decrease in beta-globin biosynthesis were observed in responsive patients. Extensive new in vivo footprints were detected in erythroblasts of responsive patients in four regions of the gamma-globin gene promoter, designated butyrate-response elements gamma 1-4 (BRE-G1-4). Electrophoretic mobility shift assays using BRE-G1 sequences as a probe demonstrated that new binding of two erythroid-specific proteins and one ubiquitous protein, alphaCP2, occurred with treatment in the responsive patients and did not occur in the nonresponder. The BRE-G1 sequence conferred butyrate inducibility in reporter gene assays. These in vivo protein-DNA interactions in human erythroblasts in which gamma-globin gene expression is being altered strongly suggest that nuclear protein binding, including alphaCP2, to the BRE-G1 region of the gamma-globin gene promoter mediates butyrate activity on gamma-globin gene expression.

Adolescent↗

PubMed1998

Differential regulation of discrete apoptotic pathways by Ras.

The products of the ras genes are known to regulate cell proliferation and differentiation; recently, they have been found to play a role in apoptosis. The expression of oncogenic p21(ras) in a number of cell types, including Jurkat (a human T lymphoblastoid cell line) and murine fibroblasts, makes the cells susceptible to apoptosis following suppression of protein kinase C (PKC) activity (PKC/Ras-mediated apoptosis). Engagement of Fas antigen, a potent effector of apoptosis, activates cellular p21(ras), which may be required for completion of the cell death program. To further investigate the role of p21(ras) in the regulation of apoptosis, the cellular mechanisms employed in these two apoptotic processes in which Ras activity is involved (PKC/Ras-related and Fas-triggered apoptosis), was explored. Increasing p21(ras) activity by expressing v-ras or by treatment with an antisense oligonucleotide to the GTPase-activating protein was found to accelerate the Fas-mediated apoptotic process in Jurkat and mouse LF cells. PKC/Ras-related apoptosis was associated with, and required, cell cycle progression, accompanied by the expression of the G1/S cyclins. In contrast, Fas engagement, although inducing a vigorous and PKC-independent activation of endogenous p21(ras), did not alter cell cycle progression, nor did it require such progression for apoptosis. Both the protein synthesis inhibitor cycloheximide and cyclin E antisense oligonucleotides partially abolished PKC/Ras-mediated apoptosis but had only a moderate effect on Fas-induced apoptosis. In contrast, the CED-3/interleukin-1beta-converting enzyme (ICE) protease inhibitor Z-VADfmk efficiently suppressed Fas-induced apoptosis and only marginally inhibited PKC/Ras-mediated apoptosis. Induction of both pathways resulted in activation of the Jun NH2-terminal kinase/JUN signaling system. These results suggest that different cell death programs, such as PKC/Ras-mediated and Fas-mediated apoptosis, may be interconnected via p21(ras) and perhaps Jun NH2-terminal kinase/JUN. In response to various death stimuli, p21(ras) may act as a common intermediate regulator in the transduction of apoptotic signals.

Animals↗

PubMed1998

Cellular and molecular effects of a pulse butyrate regimen and new inducers of globin gene expression and hematopoiesis.

Cooley's anemia is characterized by a deficiency of beta-globin chains, a relative excess of alpha-globin chains, and consequent accelerated programmed death of developing erythroid cells in the bone marrow. Increasing expression of the gamma-globin genes to adequately balance excess alpha-globin chains can ameliorate this disorder. Butyrates induce gamma-globin experimentally, but can also cause cell growth arrest with prolonged exposure or high concentrations, which in turn can accelerate apoptosis. To determine if these potentially opposing effects can be balanced to enhance therapeutic efficacy, an intermittent "pulsed" regimen of butyrate was evaluated. Following induction of gamma-globin mRNA and protein synthesis, total hemoglobin increased in beta-thalassemia patients by more than 2 g/dl above baseline, and Hb F increased above 20% in 5/8 sickle cell patients from baseline levels of 2% Hb F. Specific regulatory regions were identified in the gamma- and beta-globin gene promoters to which new binding of transcription factors, including alpha CP2 (an activator of gamma globin) occur during therapy solely in the butyrate-responsive patients. Other compounds which induce gamma globin, derivatives of acetic, phenoxyacetic, propionic, and cinnamic acids, and dimethylbutyrate, are under investigation. Some of these newer gamma-globin inducers (designed hemokines) provide better potential as therapeutics by also acting to increase hematopoietic cell viability and proliferation. Pharmacologic induction of expression of the endogenous gamma-globin genes is a realistic approach to therapy of the beta-globin disorders for many patients, with some effective agents available now and new therapeutics, with enhanced activities, under development.

Adult↗

PubMed1998

Arginine butyrate-induced susceptibility to ganciclovir in an Epstein-Barr-virus-associated lymphoma.

Lymphoproliferative disorders associated with Epstein-Barr virus (EBV) infections can occur in the setting of immunosuppression. In some patients, the lymphoproliferative disorder can resemble an aggressive monoclonal non-Hodgkins lymphoma (NHL). These NHL are poorly responsive to conventional therapy. Similarly, antiviral therapy with synthetic nucleosides such as ganciclovir are ineffective because the genes that render the virus susceptible to therapy are not expressed in EBV+ lymphomas. Using a cell line derived from a lung transplant recipient with an EBV+ immunoblastic NHL, we studied the ability of arginine butyrate to induce the expression of EBV thymidine kinase. Arginine butyrate was not only effective in inducing EBV thymidine kinase transcription, but also acted synergistically with the antiviral agent ganciclovir to inhibit cell proliferation and decrease cell viability. Based on these findings, the patient from whom the cell line was derived was treated with arginine butyrate/ganciclovir as well as conventional cytotoxic chemotherapy. No additional toxicity was observed with the arginine butyrate/ganciclovir therapy. Histologic examination of the tumor showed substantial necrosis. These observations suggest the feasibility of arginine butyrate induction of ganciclovir susceptibility in patients with EBV-associated lymphomas.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1998

Hypoxia increases thrombospondin-1 transcript and protein in cultured endothelial cells.

The exposure of endothelial cells to hypoxic environments regulates the expression of a number of genes with products that are vasoactive or mitogenic for vascular tissue, including platelet-derived growth factor, endothelin-1, and endothelial nitric oxide synthase. Hypoxia is also known to alter the adhesive properties of endothelium toward a variety of blood cell types. Thrombospondin-1 (TSP-1) is a glycoprotein with major roles in cellular adhesion and vascular smooth muscle proliferation and migration. We report here that hypoxia induces TSP-1 gene and protein expression. Oxygen tensions of < or =30 torr resulted in TSP-1 transcript induction initially apparent at 1 to 6 hours, with maximal induction (6.5-fold+/-1.2-fold) within 24 to 48 hours in both human and bovine endothelial cells. TSP-1 protein levels remain elevated after 72 hours of continuous hypoxic exposure. The induction of TSP-1 steady-state transcript levels is caused in large part, if not entirely, by post-transcriptional stabilization of the TSP-1 mRNA. The TSP-1 induction by hypoxia is a graded and reversible physiologic response and can be mimicked by the use of cobalt chloride or the inhibition of nitric oxide production, suggesting both the involvement of a heme-containing oxygen sensor and a role for the endogenous production of nitric oxide in TSP-1 regulation. The effects of hypoxia both on the stabilization of the TSP-1 transcript and the stimulation of TSP-1 protein production are completely inhibited by arginine butyrate.

Animals↗

PubMed1998

Correlation of genetic instability and apoptosis in the presence of oncogenic Ki-Ras.

The product of the ras proto-oncogene has been implicated as an essential signal transducer, involved in a variety of biological or pathological activities, including apoptosis. The aim of this investigation was to further explore the mechanisms of apoptosis triggered by Ras. Stable expression of constitutively-activated (v)-Ki-Ras in Balb/c-3T3 mouse fibroblasts resulted in a loss of G1 arrest in response to treatments which induced cell cycle arrest in the parental Balb/c-3T3 cells, accompanied by decreased expression of the p53 tumor suppressor protein and the GADD45 gene, the product of which is involved in DNA repair, and deregulated expression of the MDM-2 gene, the product of which can regulate p53 expression. Ki-Ras expression also increased the frequency of PALA-selectable CAD gene amplification, and paradoxically the susceptibility to PALA-induced apoptosis. After persistent serum-starvation, cells expressing the activated ras gene lost clonogenic potential, indicating impaired capability for genetic repair in the cells. Taken together, these data suggest that activated Ki-ras may confer genetic instabilty upon cells, possibly through interference with tumor suppressors, such as p53. While this instability may facilitate adaptation to environmental stresses, this instability in the genome also renders cells containing activated ras genes intrinsically more susceptible to programmed cell death, possibly by accumulation of undesirable or lethal genetic events during the process of tumor development.

3T3 Cells↗

PubMed1998

Butyrate-induced G1 arrest results from p21-independent disruption of retinoblastoma protein-mediated signals.

When treated with millimolar concentrations of butyrate, many cell types undergo growth arrest in the G1 phase of the cell cycle. However, the molecular basis of butyrate-induced G1 arrest has not been elucidated. We have investigated the molecular mechanisms of butyrate-induced G1 arrest in synchronized cultures of untransformed 3T3 fibroblasts. We tested the hypothesis that butyrate-induced growth arrest might be mediated by the p21 cyclin-dependent kinase inhibitor. Sodium butyrate-treated 3T3 cells did, indeed, express elevated levels of p21 mRNA under conditions of G1 arrest. Surprisingly, however, primary cultures of fibroblasts from transgenic p21 "knockout" (p21-/-) mice and fibroblasts from wild-type p21-proficient (p21+/+) mice underwent butyrate-induced G1 arrest with similar dose dependencies. Therefore, p21 expression was not necessary for butyrate-induced G1 arrest. To identify other potential mechanisms of butyrate-induced growth arrest, we analyzed the butyrate sensitivity of key mitogenic signaling events during G1. We found that butyrate inhibited the mitogen-dependent transcriptional induction of cyclin D1 and phosphorylation of retinoblastoma (Rb), both in p21-proficient 3T3 cells and in p21+/+ and p21-/- mouse embryo fibroblasts. Butyrate treatment also prevented mitogen-dependent transcriptional induction of cyclin E and expression of cyclin A, cell cycle events that are temporally distal to expression of cyclin D and are necessary for entry into S phase. Abrogation of a requirement for cyclin D/cyclin-dependent kinase-dependent phosphorylation of Rb (by ectopic expression of the human papilloma virus E7 oncoprotein in 3T3 cells) resulted in decreased sensitivity to the antiproliferative actions of butyrate. Overall, these data show that butyrate-induced G1 arrest is, in large part, independent of p21 induction. Instead, butyrate-induced growth arrest appears to result from perturbation of the Rb signaling axis at the level of or at a stage prior to cyclin D1 expression.

3T3 Cells↗

PubMed1997

A benzo[a]pyrene-induced cell cycle checkpoint resulting in p53-independent G1 arrest in 3T3 fibroblasts.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the basic helix-loop-helix family. Although physiological ligands for the AhR have not been identified, carcinogenic polycyclic aromatic hydrocarbons such as Benzo[a]pyrene (B[a]P) are high affinity AhR ligands that induce nuclear translocation and sequence-specific DNA binding of the AhR. AhR-regulated genes include members of the cytochrome P-450 family that are known to oxidize B[a]P to form genotoxic (DNA-damaging) metabolites. Murine Swiss 3T3 cells express high levels of AhR. Treatment of Swiss 3T3 cells with B[a]P during the G1 phase of the cell cycle resulted in growth arrest, as shown by inhibition of growth factor-stimulated DNA synthesis. By contrast, other murine 3T3 fibroblasts not expressing detectable levels of AhR did not undergo growth arrest in response to B[a]P. The AhR antagonist alpha-naphthoflavone prevented B[a]P-induced growth arrest, further demonstrating that cessation of cell growth was mediated by the activated AhR. A nongenotoxic AhR ligand (2,3,7,8-tetrachlorodibenzo-p-dioxin) did not elicit growth arrest, showing that ligand activation of the AhR alone was insufficient to block cell cycle progression. However, genomic DNA from B[a]P-treated Swiss 3T3 cells contained covalent adducts, whereas that from 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated cells did not, showing that G1 arrest correlated with DNA damage resulting from genotoxic B[a]P metabolites. B[a]P-induced DNA damage and growth arrest was coincident with elevated levels of nuclear p53 protein and induction of the p53-regulated mdm-2 proto-oncogene. However, Swiss 3T3 fibroblasts expressing "dominant negative" mutant p53, as well as primary fibroblasts from p53-/- "knockout" mice, also underwent growth arrest in response to B[a]P. Therefore, B[a]P-induced growth arrest occurs via p53-independent mechanisms.

3T3 Cells↗

PubMed1997

Activation of collagenase IV gene expression and enzymatic activity by the Moloney murine leukemia virus long terminal repeat.

Moloney murine leukemia virus (Mo-MuLV) is a thymotropic and leukemogenic retrovirus which causes T lymphomas and leukemias, yet does not contain a transforming gene product. Mo-MuLV has been shown to trans-activate cellular genes via a polymerase III-generated transcript, designated let, from the long terminal repeat (LTR). Here we demonstrate that introduction of the Mo-MuLV LTR stably, or transiently, into murine or human cultured cells resulted in an 8- to 15-fold increase in collagenase IV (92-kDa gelatinase, gelatinase B, matrix metalloproteinase-9) gene expression. Collagenase IV protein expression was induced 9-fold by stable integration of MuLV LTR, as measured by immunoblot analysis using an anti-collagenase IV polyclonal antibody. The MuLV LTR coordinately stimulated the proteolytic activity of collagenase IV by 14-fold. The AP-1-binding site in the collagenase IV promoter was required for transactivation by the LTR. Collagenase type IV degrades type IV collagen, a major component of basement membrane, which constitutes the first step of the metastatic cascade. The activation of proteolytic enzymes by the MuLV LTR may thus play a contributory role in the development or spread of virus-induced lymphomas or leukemias.

3T3 Cells↗

PubMed1997

Moloney murine leukemia virus long terminal repeat activates monocyte chemotactic protein-1 protein expression and chemotactic activity.

Moloney murine leukemia virus (Mo-MuLV) is a thymotropic and leukemogenic retrovirus which causes T lymphomas. Recently, Mo-MuLV has been shown to trans-activate cellular genes. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine which can promote the migration and diapedesis of monocytes and lymphocytes, as well as inducing metastasis of lymphomas. Here we demonstrate that introduction of Mo-MuLV or the MuLV LTR alone, transiently or stably, into Balb/c-3T3 cells or HeLa cells resulted in 9-11 fold increases in MCP-1 transcripts. This trans-activation of the MCP-1 gene by the Mo-MuLV LTR is independent of the physical location of the MCP-1 gene or of the LTR, occurring whether the LTR or the MCP-1 gene is integrated in the genome or transiently expressed. Immunoblot analysis using an anti-MCP-1 polyclonal antibody showed that the expression of the MuLV LTR in HeLa cells also induced the appearance of the MCP-1 protein. Boyden Chamber analysis demonstrated that the MCP-1 chemotactic activity produced by HeLa cells with an integrated MuLV LTR was elevated by 11 fold and that neutralizing antibody to human MCP-1 abrogated monocyte migration in response to MuLV LTR expression. Promoter deletional analysis showed the LTR responsive cis-acting element in the MCP-1 promoter is located between -141 and -88. Deletion of this region abolished the trans-activation of MCP-1 by the LTR. These LTR-mediated activations of a chemotactic and inflammatory cytokine may be relevant as mechanisms whereby retroviruses which do not contain oncogenes can induce neoplasia.

Animals↗

PubMed1997

Abrogation of IL-3 requirements and stimulation of hematopoietic cell proliferation in vitro and in vivo by carboxylic acids.

Short-chain fatty acids, such as butyrate and propionate, induce fetal globin gene expression and are under clinical investigation in the beta-hemoglobinopathies. Limitations of the short-chain fatty acids as therapeutics include their rapid metabolism and a tendency to induce cell growth arrest if administered for prolonged periods. In studies described here, the cellular effects of other inducers of fetal globin, phenoxyacetic acid and derivatives of short-chain fatty acids and cinnamic acids, were investigated in the human erythroid cell line K562, the IL-3 dependent multi-lineage cell line (32D), and in mice and primates. Several test compounds supported 32D cell proliferation despite a 50-fold depletion of IL-3, which resulted in growth arrest and apoptotic death in control cells. The degree of proliferation induced by certain test compounds was similar to the degree of proliferation induced by Erythropoietin and G-CSF in the cells. Eight of ten compounds induced gamma globin mRNA in K562 cells. A 2.5 to 6-fold increase in reticulocytosis was observed in vivo in mice treated with two prototype compounds. Pharmacokinetic studies of three prototype compounds demonstrated millimolar plasma concentrations after single oral doses for many hours in primates. These findings identify orally bioavailable compounds which induce gamma globin gene expression and hematopoietic cell proliferation through an activity which partially abrogates requirements for IL-3. Such compounds provide potential for oral therapeutics which stimulate proliferation of hematopoietic cells of multiple lineages, as well as inducing fetal globin.

Animals↗

PubMed1997

Microsatellite alterations indicating monoclonality in atypical hyperplasias associated with breast cancer.

One model of breast tumorigenesis postulates a sequential evolution from normal to proliferative epithelium and eventually to neoplasia, but genetic data to support this progression have been limited. We wished to determine whether atypical hyperplasia (AH), a proliferative lesion conventionally classified and treated as benign, but associated with an increased risk of developing carcinoma, might show evidence of genetic abnormalities. Using the polymerase chain reaction (PCR), we examined DNA extracted from 12 separate AH lesions, from six breast cancer patients' paraffin-embedded tissue specimens, for alterations in microsatellite repeat sequences. Five of 12 AH lesions, from three of six patients, demonstrated alterations in microsatellite sequences in patterns indicating that the AH lesions are monoclonal or contain a substantial monoclonal component. We conclude that a subset of AH lesions from patients with breast cancer are characterized by monoclonal microsatellite alterations; therefore, they may already be neoplastic. This finding lends support to one postulated sequence of breast tumorigenesis and suggests that some type of genetic instability may play a role early in breast tumor development.

Adult↗

PubMed1997

Identification of a cis-acting element in the class I major histocompatibility complex gene promoter responsive to activation by retroviral sequences.

The infection of cells with Moloney murine leukemia virus (M-MuLV) causes an increase in specific cellular gene products, including the major histocompatibility complex (MHC) class I antigens. This upregulation occurs through a transactivation process mediated by the long terminal repeat (LTR) of M-MuLV, and we show here that the gene activation response to the LTR requires at least one specific cis element within the MHC proximal promoter region. Nested deletions of MHC class I H-2Kb gene promoter sequence were subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector and then transiently introduced into BALB/c-3T3 cells expressing M-MuLV or cotransfected into BALB/c-3T3 cells with a vector containing subgenomic portions of the virus, including the LTR. CAT activity assays demonstrated that a minimal H-2Kb gene promoter (-64 to +12) contained elements sufficient for this transactivation. DNase I footprinting assays located a protein-binding site in the region of -64 to -34 bp from the transcriptional start site, and point mutation analysis confirmed the location of this cis-acting element, designated the let response element (LRE), and defined a binding motif. This LRE is distinct from binding sites for currently known transcription factors in the class I MHC gene promoter and is conserved in the promoters of human and murine MHC class I genes. Mutation of the LRE resulted in dramatic reduction in both DNA-protein binding activity in electrophoretic mobility shift assay and in the ability of the mutated promoter to respond to retroviral transactivation. Addition of the LRE to a heterologous promoter conferred the ability to respond to retroviral transactivation.

3T3 Cells↗

PubMed1997

Protection from apoptosis in human neutrophils is determined by the surface of adhesion.

Recent work suggests that various neutrophil agonists affect the rate of apoptosis in these cells. On the basis of these observations, we hypothesized that signals triggered in neutrophils via their adhesion receptors might also modify their life span. This hypothesis has been tested using human neutrophils adherent to tissue culture plastic, either untreated or coated with extracellular matrix (ECM) proteins or with monolayers of human umbilical vein endothelial cells. To detect and quantitate apoptotic changes in adherent cells, we developed a microtiter plate assay using a cell-permeable DNA-binding fluorescent dye, Hoechst 33342. Use of this assay demonstrated that 1) the number of apoptotic cells among neutrophils adherent to plastic after 6-20 h of incubation was significantly lower than that among neutrophils adherent to the ECM proteins fibronectin or laminin; 2) adhesion to interleukin-1-activated endothelial cells delayed apoptosis, whereas adhesion to nonactivated endothelium accelerated neutrophil death; and 3) monoclonal antibodies directed against intercellular adhesion molecule 1 or against the common beta 2-chain of the leukocyte integrins abolished the protective effect of interleukin-1-activated endothelial cells on apoptosis of adherent neutrophils. These results suggest that the life span of adherent neutrophils. depends on the activating signals triggered by the surface of adhesion.

Apoptosis↗

PubMed1997

Regulation of c-jun gene expression in endothelial cells by the protein kinase inhibitor staurosporine.

The proto-oncogene c-jun, a member of the family of immediate-early genes, is transcriptionally induced in different cell types by a variety of stimuli, including mitogens, tumor promoters, growth factors. We show here that the protein kinase inhibitor staurosporine, which inhibits both the serine-threonine and tyrosine specific protein kinases, also causes differential regulation of the c-jun gene in endothelial cells. Increasing concentrations of staurosporine modulated the steady-state levels of c-jun mRNA in bovine aortic endothelial (BAE) cells in a multiphasic manner. The half-life of c-jun mRNA did not change significantly under these conditions, suggesting that the modulations in the mRNA levels were caused primarily by differential transcriptional activity of the gene. The expression of c-jun gene is believed to be regulated by its own product, the JUN protein, which constitutes a major component of the inducible transcription factor AP-1. In order to test whether the differential regulation of c-jun gene was caused by the differential activation (or inactivation) of the AP-1 transcription factor, the DNA-binding activity of this transcription factor in staurosporine-treated cells was measured. Gelshift analysis with a synthetic oligonucleotide probe showed modest effects of staurosporine on the DNA-binding activity of the transcription factor AP-1. The changes observed in the DNA-binding activity of AP-1 did not parallel the changes observed in the steady-state levels of c-jun mRNA. Similarly, the expression of an AP-1 dependent reporter gene construct was regulated in a fashion entirely different from the c-jun gene during the same protein kinase inhibitory conditions. These results suggest the existence of an alternative pathway that regulates the c-jun gene expression in endothelial cells independent of both the protein kinase and AP-1 transcription factor activation steps.

Animals↗