Clonal evolution of N-methylnitrosourea-induced C57BL/6J thymic lymphomas by analysis of multiple genetic alterations.
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Animals↗
Biomedical subjects
J W Ludlow
Publications and source records attributed to J W Ludlow.
Hypoxia arrests ovarian carcinoma cell cycle progression, but invasion is unaffected.
Although hypoxic cells are generally resistant to radiation and chemical therapies designed to halt the spread of neoplastic disease, few investigations have been carried out with regard to the molecular mechanisms responsible for this phenomenon. Here, we report of the development of an in vitro model system with which to study the molecular mechanisms involved in the proliferation and invasion of human ovarian carcinoma cells under hypoxia. Results from [(3)]thymidine incorporation experiments indicate that hypoxia triggers cessation of ovarian carcinoma cell DNA synthesis. Flow cytometry analysis of cellular DNA content for hypoxic cultures revealed that cell cycle progression was arrested. This arrest was found to be reversible upon reoxygenation of the cultures. Concomitant with this growth arrest is hypophosphorylation of pRB and a reduction in cyclin A abundance, suggesting that hypoxia induces growth arrest by regulating the activities of these crucial cell cycle-regulatory proteins. In vitro invasion assays revealed that hypoxia has no appreciable effect on the invasive ability of these cells. Immunoblotting established that the detected proteolytic activity was due to the matrix metalloproteinase MMP-2, the M(r) 72,000 type IV collagenase that is most closely associated with the metastatic phenotype in vitro and in vivo. These data support the notion that populations of ovarian carcinoma cells are capable of surviving and invading extracellular matrix during hypoxic conditions and, after a more suitable oxygen environment is reached, giving rise to new cell colonies.
Carcinoma↗
Use of sequential immunoprecipitation to reveal discrete, separable populations of SV40 T-antigen binding to host cellular proteins.
Sequential immunoprecipitations were carried out to determine the usefulness of this method for separating subpopulations of SV40 T-antigen complexed to various combinations of the cellular growth regulatory proteins pRB, p107, and p53. This approach was used successfully to separate discrete populations of SV40 T-antigen in a quatramolecular complex with pRB, p53, and p107, a trimolecular complex with pRB and p107, and a trimolecular complex with p107 and p53. This method was used as the first step towards isolating T-antigen for subsequent phosphopeptide mapping to address whether alterations in the overt phosphorylation of this viral oncoprotein is a major determinating factor to separation of T-antigen populations by complexing with different combinations of cellular growth regulatory proteins.
Animals↗
Association of p300 and CBP with simian virus 40 large T antigen.
p300 and the CREB-binding protein CBP are two large nuclear phosphoproteins that are structurally highly related. Both function, in part, as transcriptional adapters and are targeted by the adenovirus E1A oncoprotein. We show here that p300 and CBP interact with another transforming protein, the simian virus 40 large T antigen (T). This interaction depends on the integrity of a region of T which is critical for its transforming and mitogenic properties and includes its LXCXE Rb-binding motif. T interferes with normal p300 and CBP function on at least two different levels. The presence of T alters the phosphorylation states of both proteins and inhibits their transcriptional activities on certain promoters. Although E1A and T show little sequence similarity, they interact with the same domain of p300 and CBP, suggesting that this region exhibits considerable flexibility in accommodating diverse protein ligands.
Amino Acid Sequence↗
Tumour suppressor genes in disease and therapy.
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Cell Division↗
Effect of the kinase inhibitor 2-aminopurine on the cell cycle stage-dependent phosphorylation state of the retinoblastoma protein.
We investigated the effect of 2-aminopurine (2-AP) on the cell cycle stage-dependent phosphorylation state of the retinoblastoma growth suppressor protein (pRB). Our results suggest that 2-AP does not affect dephosphorylation of pRB scheduled during metaphase/anaphase of mitosis, even though 2-AP-treated cells never enter metaphase or anaphase. Phosphorylation of pRB, which is important for G1 exit, appeared to be impeded by 2-AP late during G1, coincident with a slowing of G1 cell progression into S phase. The significance of these data with regard to cell cycle regulation is discussed.
2-Aminopurine↗
Control and activity of type-1 serine/threonine protein phosphatase during the cell cycle.
The first part of this discourse will serve as a compendium of selected aspects of PP1 activity and control during the cell cycle. The latter sections will focus on the mammalian cell cycle dependent activity of PP1 on the negative growth regulatory product of the retinoblastoma susceptibility gene, pRB. The purpose here is to suggest one possible mechanism to explain how this phosphatase can effect cell cycle progression. An argument is made for PP1 playing an indirect role in regulating cell cycle progression by modulating the growth suppressive activity of pRB.
Animals↗
Viral oncoprotein binding to pRB, p107, p130, and p300.
The purpose of this review is to bring attention to some additional work in the tumor virus/tumor suppressor field which may have been overshadowed by reports describing adenovirus, SV40, and HPV oncoprotein binding to pRB and p53. The data reviewed herein provide further support for the model that a common mechanism by which DNA tumor viruses transform cells involves inactivation of cellular proteins which function as negative regulators of cell growth.
Animals↗
Prostaglandin E2 inhibits B lymphocyte activation by a cAMP-dependent mechanism: PGE-inducible regulatory proteins.
We have previously shown that macrophage-secreted prostaglandins of the E series (PGE) and other agents which increase cAMP inhibit IgM production and proliferation of murine B lymphocytes. In this study, we show that PGE2 inhibits B cell activation events including enlargement, class II MHC hyperexpression, and the expression of the low-affinity receptor for IgE, Fc epsilon RII/CD23 (35-50%) in a cAMP-dependent manner. PGE action is mimicked by other cAMP-inducing agents and is inhibited by RpcAMP (a nonhydrolyzable cAMP analog which is a competitive inhibitor of cAMP-dependent protein kinase A). PGE2 could inhibit enlargement and upregulation of activation Ag even if preincubated with cells and then washed out prior to B cell stimulation. This change in B cell phenotype was abrogated if the reversible protein synthesis inhibitor cycloheximide was included during B cell incubation with PGE2. To identify the newly synthesized cAMP- and PGE-inducible regulatory proteins (PIRP), two-dimensional gel electrophoresis of lysates of B lymphocytes treated +/- PGE2 was performed. This report is the first to identify putative PIRP proteins. The roles of PIRP in PGE regulation of B cell activation and class switching are discussed.
Animals↗
Interactions between SV40 large-tumor antigen and the growth suppressor proteins pRB and p53.
The oncogenic property of simian virus 40 depends in large part on the function of the virus-coded T-antigen. Although the precise mechanism of how T functions during neoplastic transformation is not clear, some answers to this question may lie in our understanding the nature of the proteins found to complex with T. The cellular protein p53 is perhaps the most extensively studied protein in this regard. Recently, p53 was defined as a growth suppressor protein. At about this same time, T was found to complex with another cell growth suppressor protein, the product of the retinoblastoma susceptibility gene. It has since become apparent that complex formation between these proteins affects their individual growth-modulating activities. Quite often this alteration of activity correlates with an uncontrolled proliferative state of the cell. Thus, transformation by SV40 is thought to involve complex formation between the viral T oncoprotein and cellular growth suppressor proteins. This complex formation is believed to result in nullification of the growth suppressor protein properties, thus increasing the propensity of the cell toward uncontrolled growth, the hallmark of neoplastic transformation.
Animals↗
Specific enzymatic dephosphorylation of the retinoblastoma protein.
The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W. Ludlow, J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio, Cell 60:387-396, 1990). To determine the precise timing of RB dephosphorylation and whether a specific phosphatase is active in this process, we have utilized a nocodazole block and release protocol which allows a large population of cells to progress synchronously through mitosis. In such experiments, RB dephosphorylation began during anaphase and continued until complete dephosphorylation was apparent in the ensuing G1 period. In addition, late mitotic cell extracts were capable of dephosphorylating RB in vitro. This RB-specific mitotic phosphatase activity was more active in anaphase extracts than in pro- or metaphase extracts, which is consistent with the results obtained in vivo. Okadaic acid and protein phosphatase inhibitors 1 and 2 inhibited this specific RB phosphatase activity. These results suggest a role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB.
Animals↗
Hypoxic stress induces reversible hypophosphorylation of pRB and reduction in cyclin A abundance independent of cell cycle progression.
The effect of hypoxic stress on the phosphorylation state of the product of retinoblastoma susceptibility gene (pRB) and cyclin A abundance was examined in CV-1P monkey kidney cells. Flow cytometric DNA histogram analysis and [3H]-thymidine incorporation assays demonstrated that hypoxia inhibited cell cycle progression and cell division. Within 6-12 h of hypoxia, pRB became hypophosphorylated and cyclin A abundance fell below detection limits. Hypophosphorylation of pRB and loss of cyclin A detection occurred without progression of cells through S-phase. These effects were found to be reversible by reoxygenation of the hypoxic cultures. Cells were shown to resume DNA synthesis within 12-16 h of reoxygenation concomitant with pRB hyperphosphorylation and an increase in cyclin A detection. These data demonstrate that hypoxic stress blocks the progression of these cells through the phases of the cell cycle and suggests that the effect might arise from the down regulation of key cell cycle controlling elements. The data also raise the possibility that maintaining pRB in a hyperphosphorylated state may be crucial for S-phase progression as well as S-phase entry.
Animals↗
Regions of human papillomavirus type 16 E7 oncoprotein required for immortalization of human keratinocytes.
Binding of the retinoblastoma gene product (pRB) by viral oncoproteins, including the E7 of human papillomavirus type 16 (HPV 16), is thought to be important in transformation of cells. One of the steps in transformation is the immortalization process. Here we show that mutations in E7 within the full-length genome which inhibit binding of pRB do not abrogate the ability of the HPV 16 DNA to immortalize primary human epithelial (keratinocyte) cells. A mutation in one of the cysteines of a Cys-X-X-Cys motif which is contained in the carboxy half of the E7 and is part of a zinc finger arrangement completely eliminates the ability of HPV 16 DNA to immortalize cells. The results indicate the importance of E7 in the immortalization of primary keratinocytes but suggest that the binding of pRB is not essential.
Amino Acid Sequence↗
Selective ability of S-phase cell extracts to dephosphorylate SV40 large T antigen in vitro.
Simian virus 40 (SV40) large tumor antigen (T) is an oncoprotein whose biological and biochemical functions appear to be modulated by phosphorylation. Recently, SV40 DNA replication in vitro has been shown to be activated by dephosphorylation involving the activity of a serine/threonine phosphoprotein phosphatase belonging to the type 2A class (PP2A) [Virshup, D.M., Kauffman, M.G. & Kelly, T.J. (1989) EMBO J., 8, 3891-3898]. To address the question of how specificity of PP2A activity towards T is regulated, an in vitro assay to study the process of T dephosphorylation was developed. Unlabeled extracts from cells enriched for various stages of the cell cycle were incubated with 32P-labeled, immunocomplexed T. Extracts from a population of cells enriched for S phase demonstrated a selective ability to dephosphorylate this labeled protein when compared with extracts prepared from G1- and M-phase cells. The time course of release from growth arrest demonstrated that this T-specific phosphatase activity occurred at the onset of host-cell DNA synthesis. In contrast, when using 32P-labeled phosphorylase a as the substrate, phosphatase activity appeared to be present throughout the cell cycle. The data presented here are consistent with the notion that PP2A activity towards T is regulated in a cell cycle-dependent manner.
Antigens, Viral, Tumor↗
Anti-immunoglobulin treatment of murine B-cell lymphomas induces active transforming growth factor beta but pRB hypophosphorylation is transforming growth factor beta independent.
Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, leading to apoptosis and cell death in the immature lymphomas WEHI-231 and CH31, but not in the CH12 B-cell line. In this system, which has been used as a model for B-cell tolerance, we have established that these lymphomas produce active transforming growth factor beta (TGF-beta) when treated with anti-Ig and that their hierarchy of sensitivity to TGF-beta generally correlates with their growth inhibition by anti-Ig. TGF-beta, in turn, has been shown to interfere with the phosphorylation of the retinoblastoma gene product, pRB. Herein, we also demonstrate that in WEHI-231 B-lymphoma cells treated with anti-Ig for 24 h, the pRB protein is found to be predominantly in the underphosphorylated form, as previously reported for cells arrested by the exogenous addition of TGF-beta. However, neutralizing antibodies to TGF-beta failed to prevent growth inhibition by anti-Ig in WEHI-231 and CH31. When WEHI-231 lymphoma cells were selected for growth in TGF-beta, the majority of the TGF-beta-resistant clones remained sensitive to anti-Ig-mediated growth inhibition. In these clones, the retinoblastoma gene product was found to be in the underphosphorylated form after 24-h treatment with anti-Ig, but not with TGF-beta. These data show that anti-Ig treatment of murine B-cell lymphomas stimulates the production of active TGF-beta but that a TGF-beta-independent pathway may be responsible for the pRB underphosphorylation and cell cycle blockade.
Animals↗
Control of junB and extracellular matrix protein expression by transforming growth factor-beta 1 is independent of simian virus 40 T antigen-sensitive growth-sensitive growth-inhibitory events.
Treatment of Mv1Lu mink lung epithelial cells with transforming growth factor-beta 1 (TGF-beta 1) prevents phosphorylation of the retinoblastoma susceptibility gene product, RB, in late G1 phase of the cell cycle, which is thought to retain RB in a growth-suppressive state. This effect is paralleled by cell cycle arrest in late G1 (M. Laiho, J. A. DeCapric, J. W. Ludlow, D. M. Livingston, and J. Massagué, Cell 62:175-185, 1990). Arrest can be prevented by expression of simian virus 40 T antigen, which binds to underphosphorylated RB, presumably blocking its growth-suppressive activity. The response of cells to TGF-beta 1, however, is complex and includes changes in the levels of expression of genes encoding nuclear transcription factors and extracellular matrix components. To define the relationships among various components of the TGF-beta 1 response, we have investigated the effect of TGF-beta 1 on cells whose growth-inhibitory response to this factor is prevented by T antigen. TGF-beta 1 addition to exponentially growing Mv1Lu cells increased the levels of junB mRNA and of three extracellular matrix proteins: plasminogen activator inhibitor-1, fibronectin, and thrombospondin. Kinetically, the effects on junB and plasminogen activator inhibitor-1 expression occurred faster (half-maximal at 1 to 2 h) than the effects on fibronectin and thrombospondin expression (half-maximal at 6 to 10 h). These effects either preceded or overlapped, respectively, the withdrawal of Mv1Lu cells from the cell cycle. Expression of a transfected T-antigen gene in Mv1Lu cells, however, did not prevent any of these responses to TGF-beta 1. The results indcate that TGF-B1-stimulated expression of junB and extracellular matrix proteins in Mv1Lu cells can occur independently of the T-antigen-sensitive events that lead to growth arrest.
Animals↗
Growth inhibition by TGF-beta linked to suppression of retinoblastoma protein phosphorylation.
The growth-suppressive function of the retinoblastoma gene product, RB, has been ascribed to the underphosphorylated RB form that prevails during G1 phase in the cell cycle. We show that addition of the paracrine growth inhibitor transforming growth factor beta 1 (TGF-beta 1) to Mv1Lu lung epithelial cells in mid to late G1 prevents phosphorylation of RB scheduled for this cell cycle stage and arrests cells in late G1. Expression of SV40 T antigen, a transforming protein that binds underphosphorylated RB, does not block the effect of TGF-beta 1 on RB phosphorylation but greatly reduces the growth-inhibitory response to TGF-beta 1. TGF-beta 1 and RB appear to function in a common growth-inhibitory pathway in which TGF-beta 1 acts to retain RB in the underphosphorylated, growth-suppressive state.
Animals↗
The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T.
Synchronized monkey cells pulse-labeled with [35S]-methionine and chased for various lengths of time were extracted, and immunoprecipitations were performed using monoclonal antibodies directed against the retinoblastoma protein (RB) and SV40 T antigen (T). By following a discrete population of these two proteins through the cell cycle, the following information was obtained. RB, which is wholly unphosphorylated in G1, became phosphorylated at the beginning of S and remained phosphorylated through S and G2. RB was, then, completely dephosphorylated between the end of G2 and the beginning of G1. Second, while all of the detectable unphosphorylated RB can be found complexed with T, these complexes present during G1 dissociated in S and reformed again in M or early G1. Finally, T molecules appeared to oligomerize prior to binding RB. Thus, complex formation between T and RB may be regulated in part by the cell cycle-dependent phosphorylation and dephosphorylation of RB and by the quaternary structure of T.
Animals↗