The spectrum of mutations at the p53 locus. Evidence for tissue-specific mutagenesis, selection of mutant alleles, and a "gain of function" phenotype.
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Alleles↗
Biomedical subjects
J Lin
Publications and source records attributed to J Lin.
Cell cycle gene expression and E2F transcription factor complexes in human melanoma cells induced to terminally differentiate.
Defects in cellular differentiation are a common occurrence in human cancers. The combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound mezerein (MEZ) results in an irreversible loss of proliferative capacity and terminal cell differentiation in H0-1 human melanoma cells. In contrast, either agent alone induces reversible growth arrest and/or specific components of the differentiation process without inducing terminal differentiation. The current study investigates changes in cell cycle, cell cycle gene expression and E2F transcription factor complex formation during the processes of reversible and irreversible (terminal) differentiation. Induction of both terminal differentiation and reversible differentiation (MEZ treatment) results in a temporal decrease in DNA synthesis and the percentage of cells in S phase and a decrease in the expression of cell cycle and growth regulated genes, including cdc2, cyclin A, cyclin B, histone H1, histone H4, nm23-H1, p53 and c-myc. Persistent gene expression changes occur in terminally differentiated cells, but not in reversibly differentiated cells. H0-1 cells contain several E2F binding activities, including uncomplexed E2F, an E2F-p107-cyclin A-cdk2 kinase complex and an Rb-E2F complex. Induction of growth arrest by MEZ results in a slow migrating gelshift band that contains E2F associated with the pRb2/p130 protein. There is also a loss of the Rb-E2F complex. Induction of terminal differentiation after treatment with IFN-beta + MEZ generates a second pRb2/p130-E2F complex that migrates considerably faster than the pRb2/p130-E2F complex resulting from growth arrest. The slower migrating complex may contribute to growth arrest, whereas the faster migrating complex may play a role in terminal differentiation. Our results demonstrate that terminal cell differentiation involves a co-ordinate and continuous suppression of a number of cell cycle and growth related genes and results in the development of a novel E2F transcription factor complex not apparent in growth arrested and reversibly differentiated human melanoma cells.
CDC2 Protein Kinase↗
Potentiation of B-cell antigen receptor-mediated signal transduction by the heterologous src family protein tyrosine kinase, src.
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Animals↗
Essential role for p53-mediated transcription in E1A-induced apoptosis.
Baby rat kidney (BRK) cell lines transformed by E1A and a temperature-sensitive p53 [tsp53(val135)] undergo rapid apoptosis when p53 assumes the wild-type conformation at the permissive temperature. Wild-type p53 function is therefore required for induction of apoptosis in response to growth deregulation by E1A. BRK cells transformed by E1A and a transcriptionally defective temperature-sensitive p53 [tsp53(22-23val135)] are dramatically impaired for the ability to mediate E1A-induced apoptosis at the permissive temperature. The tsp53(22-23val135), however, still retains some ability to suppress cell growth. Thus, the activity of p53 as a transcription factor is directly correlated with the ability of E1A to induce apoptosis. In addition, there may exist at least two different mechanisms by which p53 can suppress cell-cycle progression, only one of which is dependent on p53-mediated transcription.
Adenovirus E1A Proteins↗
Hispanic versus white smoking patterns by sex and level of education.
Although past studies have compared cigarette smoking patterns in Hispanics and whites, few have examined differences within sex and educational subgroups. Data are presented for 1,088 Hispanic women and men (89% Mexican-American origin) and pairwise matched white women and men (544 pairs), aged 25-74 years, who participated in population-based cross-sectional surveys in California in 1979-1990. Each pair was matched on age, sex, educational level, city of residence, and survey time period. There were large differences in smoking prevalence rates between Hispanic and white pairs with low educational attainment. White women and men with less than a high school education were approximately twice as likely to be current daily cigarette smokers as were similarly educated Hispanic women and men (46.1 vs. 20.6% for women and 52.7 vs. 30.1% for men). As the level of education increased, these ethnic differences in smoking decreased and became negligible among those who completed college. Virtually all low-educated white men (92.5%) and most low-educated white women (73.1%) were either current or former daily smokers. There were large ethnic differences in rates of smoking cessation advice from a physician; only 8.3% of low-educated Hispanic men who were current daily smokers had ever been advised by a physician to stop smoking, compared with 59.6% of low-educated white men. These data confirm ethnic differences in smoking behavior and identify the high smoking rates of white men and women with low educational attainment, thus delineating an often unrecognized group toward whom tobacco prevention and cessation activities should be directed.
Adolescent↗
Transforming growth factor beta 1 costimulated growth and regulatory function of staphylococcal enterotoxin B-responsive CD8+ T cells.
Transforming growth factor beta (TGF-beta) exhibits diverse effects on growth and differentiation of a wide range of cell types. In the immune system, TGF-beta 1 is a potent inhibitor of T cell proliferation and certain T cell effector functions. However, TGF-beta 1 also enhances growth of T cells, predominantly of naive phenotype, and induces their expression of selected cytokines. We have previously demonstrated that TGF-beta 1 costimulates growth of highly purified murine CD8+ T cells activated by immobilized anti-CD3 Ab. TGF-beta 1-costimulated CD8+ T cells rapidly express a memory phenotype, lose lytic function, and express a mixed cytokine pattern with IL-2, IFN-gamma, and appreciable IL-10, as well as TGF-beta 1. The present work examines the possibility that TGF-beta 1 similarly costimulates response of murine CD8+ T cells to the microbial superantigen staphylococcal enterotoxin B (SEB) and characterizes their effector and regulatory functions. TGF-beta 1 significantly enhances CD8+ T cell proliferation to SEB in the presence of MHC class II-positive APC and TGF-beta 1-primed CD8+ T cells are enriched for SEB-reactive V beta 8+ TCR expression. TGF-beta 1 priming also up-regulates a memory-like CD45RBlowCD44highMEL-14low phenotype. TGF-beta 1 priming inhibits development of SEB-specific lytic effector function by more than 90%. However, TGF-beta 1-primed CD8+ effector T cells express elevated levels of IL-10 and TGF-beta 1, variable IFN-gamma, and undetectable IL-4. Additionally, they exhibit growth inhibitory effector function of SEB-induced proliferation of other CD4+ and CD8+ T cells. Growth inhibition by TGF-beta 1-primed CD8+ T cells is reversed in part by anti-IL-10 Ab. Thus, in the context of SEB response, TGF-beta 1 promotes the outgrowth and induces the effector function of CD8+ T cells that have the capacity to impair T cell clonal growth.
Animals↗
A role in B cell activation for CD22 and the protein tyrosine phosphatase SHP.
CD22 is a membrane immunoglobulin (mIg)-associated protein of B cells. CD22 is tyrosine-phosphorylated when mIg is ligated. Tyrosine-phosphorylated CD22 binds and activates SHP, a protein tyrosine phosphatase known to negatively regulate signaling through mIg. Ligation of CD22 to prevent its coaggregation with mIg lowers the threshold at which mIg activates the B cell by a factor of 100. In secondary lymphoid organs, CD22 may be sequestered away from mIg through interactions with counterreceptors on T cells. Thus, CD22 is a molecular switch for SHP that may bias mIg signaling to anatomic sites rich in T cells.
Amino Acid Sequence↗
Two critical hydrophobic amino acids in the N-terminal domain of the p53 protein are required for the gain of function phenotypes of human p53 mutants.
Some mutant forms of the p53 protein have been shown to gain new functions that are not shared by the wild-type p53 protein; (1) mutant p53 proteins can transcriptionally transactivate the multi-drug resistance gene-1 (MDR-1) and (2) when expressed in non-tumorigenic cells with no endogenous p53 protein, mutant p53 proteins can enhance the tumorigenic potential of these cells (Dittmer et al., 1993). It has recently been shown (Lin et al., 1994b) that the transcriptional activator domain of the p53 protein contains two amino acids, leu-22 and trp-23, which are required by the wild-type p53 protein for transcriptional activity. To determine whether these same amino acid residues are utilized by mutant p53 proteins for their gain of function phenotype, the triple mutant p53 protein (at residues 22 and 23 in the transactivation domain and residue 281 in the DNA binding domain--a gain of function mutant) was made. While the p53-281 mutant transcriptionally activates the MDR-1 gene and enhances the tumorigenic potential of cells it is expressed in, the 22, 23, 281 triple mutant failed to carry out either of these functions.
Animals↗
Electron transfer from cytochrome c to 8-azido-ATP-modified cytochrome c oxidase.
Bovine heart cytochrome c oxidase (CcO) has been modified by 8-azido-adenosine 5'-triphosphate (8-azido-ATP), and the electron-transfer activity from ferrocytochrome c to the modified CcO under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The kinetics of intermolecular electron transfer between the redox protein partners was shown to be reduced significantly. In addition, there is significant decrease in the binding affinity of the cytochrome c to the oxidase upon 8-azido-ATP modification. The 8-azido-ATP-modified CcO exhibited 50% of the intracomplex electron-transfer rate (ket) and 56% of the association constant (Ka) normally observed between cytochrome c and native CcO under otherwise identical conditions. Since the effective electron transfer rate constant is the product of ket and Ka under nonsaturation conditions, the overall electron-transfer rate has been curtailed by over a factor of 2. Similar observations have been noted with the native CcO in the presence of 3 mM ATP. In contrast, the redox potential of neither CuA nor cytochrome a was altered upon 8-azido-ATP modification or in the presence of 3 mM ATP. Also, no gross structural changes at either the CuA or the cytochrome a site were noted, as evidenced by a lack of any spectral perturbations in the EPR signals from both of these centers. We conclude that ATP modulates the electron transfer from cytochrome c to CcO by interacting with the CcO and altering allosterically the docking. In this manner, ATP can affect the branching of the electron input from ferrocytochrome c to cytochrome a and CuA.
Adenosine Triphosphate↗
The melanoma differentiation-associated gene mda-6, which encodes the cyclin-dependent kinase inhibitor p21, is differentially expressed during growth, differentiation and progression in human melanoma cells.
The combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound mezerein (MEZ) induces terminal differentiation with an irreversible loss of proliferative capacity in human melanoma cells. Using subtraction hybridization, cDNAs were identified that display enhanced expression in terminally differentiated and growth arrested human melanoma cells (Jiang and Fisher, 1993; Jiang et al., 1994a). A specific melanoma differentiation-associated (mda) cDNA, mda-6, is described whose expression inversely correlates with melanoma progression and growth. mda-6 is identical to WAF1/CIP1/SDI1 that encodes the M(r) 21,000 protein (p21) that is an inhibitor of cyclin-dependent kinases. Actively growing normal melanocyte, SV40-immortalized human melanocyte and dysplastic nevus cell lines synthesize elevated levels of mda-6 mRNA; whereas, actively proliferating radial and early vertical growth phase primary melanomas as well as metastatic human melanoma cells produce reduced levels of mda-6 mRNA. Treatment of primary and metastatic human melanoma cells with IFN-beta + MEZ results in growth inhibition and an increase in mda-6 expression. mda-6 expression also increases when human melanoma cells are grown to high saturation densities or when grown in serum-free medium. Using anti-p53 and anti-p21 antibodies, an inverse correlation is found between p53 and p21 protein levels during growth arrest and differentiation. Induction of growth arrest and terminal differentiation in H0-1 human melanoma cells by IFN-beta + MEZ results in a temporal decrease in wild-type p53 protein levels with a corresponding increase in p21 levels. In the Matrigel-assisted melanoma progression model, mda-6 expression decreases in early vertical growth phase primary human melanoma cells selected for autonomous or enhanced tumor formation in nude mice. In metastatic human melanoma cells displaying a loss of metastatic potential resulting from introduction of a normal human chromosome 6, mda-6 mRNA levels increase. Taken together, these studies indicate that mda-6 (p21) may function as a negative regulator of melanoma growth, progression and metastasis.
Amino Acid Sequence↗
Lymphocytes from pregnant women express human chorionic gonadotropin/luteinizing hormone receptor gene.
Previous studies have indicated that human chorionic gonadotropin (hCG) has immunoregulatory properties and alters the functions of human lymphocytes. However, it has not been determined whether the gene encoding the receptors for hCG/luteinizing hormone (LH) is expressed in human lymphocytes. Total peripheral mononuclear lymphocytes isolated from blood samples of pregnant women by Ficoll-Paque gradient centrifugation contained mRNA transcripts encoding the hCG/LH receptors and a 50 kDa receptor protein which can bind 125I-hCG. T-Lymphocytes isolated from total mononuclear cell fractions also contained these receptor mRNA transcripts as well as the receptor protein. The levels of receptor transcripts and receptor protein are lower in lymphocytes than in ovarian tissue. These findings suggest that the immunoregulatory actions of hCG are probably mediated by specific receptors in T-lymphocytes from pregnant women.
Blotting, Western↗
Anti-CD2 monoclonal antibody-induced receptor changes: down modulation of cell surface CD2.
Anti-CD2 mAbs suppress T cell immunity and prolong allograft survival in vivo while inducing the down-modulation of CD2 expression. Manipulation of cell surface molecules may be important in inducing tolerance, so down-modulation of CD2 expression on T cells by anti-CD2 mAbs was further defined with an in vitro model. The anti-CD2 mAb 12-15 caused CD2 expression on purified splenic T cells to decrease from 83.4 to 22.7% total positive cells while CD3, CD4, and CD8 expression remained unchanged. The expression of other adhesion molecules, LFA-1 alpha (CD11a), LFA-1 beta (CD18), Pgp-1 (CD44), CD45, MEL-14 (L-selectin), and VLA-4 alpha (CD49d), were all increased as a result of anti-CD2 treatment, whereas CD25 (IL-2R), CD48 (CD2 ligand), and ICAM-1 (CD54) remained unchanged. Kinetics showed that CD2 down-modulation was persistent and at the same magnitude from day 1 through day 7 of culture. Anti-CD2 mAb could down modulate CD2 on both CD4 and CD8 splenic lymphocyte subsets, thymocytes, and the T cell lymphoma EL-4; and, non-T cells did not seem to participate in the process of modulation. Mechanistic studies of mAb action showed that, in addition to 12-15, other anti-CD2 mAbs could cause down-modulation of T cell CD2 expression in an epitope and isotype dependent fashion and that CD2 down-modulation correlated with inhibition of receptor-driven T cell stimulation. Intact antibody, including the Fc portion, was required to induce CD2 down-modulation, and additional experiments suggested an interaction with an Fc gamma R other than Fc gamma RII or Fc gamma RIII. CD2 down-modulation did not change with the addition of the cytokines IL-1, IL-2, IL-6, IL-10, TNF alpha, or TGF-beta 1. These results show that anti-CD2 mAbs significantly and persistently down-modulate CD2 on various T cell subpopulations. The mAbs must interact with both the CD2 receptor and an Fc gamma R. CD2 down-modulation is accompanied by changes in the array of other T cell surface receptors that may contribute to mechanisms of anti-CD2-induced immunosuppression.
Animals↗
Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immunosuppression with TGF-beta 1 or vIL-10.
The application of gene transfer techniques to organ transplantation offers the potential for modulation of immunity directly within an allograft without systemic side effects. Expression vectors and promoter elements are important determinants of gene transfer and expression. In this study, various vectors (naked plasmid DNA, retroviral vector, herpes simplex viral vector, and adenoviral vector) with various promoters (RSV-LTR, SV40, MuLV-LTR, HCMVie1) were directly compared to demonstrate the successful gene transfer and expression of beta-galactosidase in murine myoblasts in vitro and within murine heterotopic, nonvascularized cardiac isografts or allografts in vivo. Expression of transferred genes was not toxic to cells and strength of expression varied according to the type of vector. Plasmid DNA was expressed in myocytes, retroviral vector was expressed in the graft infiltrating cells, and herpes simplex and adenoviral vectors were expressed in both myocytes and graft-infiltrating cells. Preliminary studies evaluated the ability of these vectors to deliver immunologically important signals. Allografts injected with pSVTGF-beta 1, a plasmid-encoding transforming growth factor beta 1 (TGF-beta 1) under the control of the SV40 promoter, showed significant prolongation of graft survival of 26.3 +/- 2.5 days compared with 12.6 +/- 1.1 days for untreated allografts, and 12.5 +/- 1.5 days for the allografts injected with control plasmid (P < 0.05). Allografts injected with MFG-vIL-10, a retroviral vector encoding viral interleukin-10 under the control of the MuLV-LTR, showed prolongation of graft survival of 36.7 +/- 1.3 days versus 12.6 +/- 1.1 days for the untreated allograft, and 13.5 +/- 2.0 days for the allografts injected with control retroviral vector (P < 0.001). Both vectors were transcriptionally active in vivo and did not appear to have toxic effects. Gene therapy for transplantation can induce transient expression of immunologically relevant molecules within allografts that impede immune activation while avoiding the systemic toxicity of conventional immunosuppression.
Animals↗
A novel integration signal that is composed of two transmembrane segments is required to integrate the Neurospora plasma membrane H(+)-ATPase into microsomes.
The Neurospora plasma membrane H(+)-ATPase belongs to a family of cation-motive porters called P-type ATPases. Putative transmembrane segments of these enzymes contain one or more charged residues. Conditions were determined by which a transmembrane segment with charged residues is integrated into its cognate membrane. We constructed fusion proteins flanked by the hydrophilic domains of the amino and carboxyl termini of the H(+)-ATPase that contained either one or two transmembrane segments. Neurospora in vitro translation system supplemented with homologous microsomes was programmed with RNA transcripts of these constructs. When transmembrane segment number one (M1) or number two (M2) of the H(+)-ATPase was engineered into the construct, the resultant protein did not integrate into microsomes. When M1 and M2 were placed in tandem, the resultant protein integrated into microsomes as judged by the criteria of resistance to extraction at pH 11.5 and protection from protease digestion. The integration event depended on ATP and GTP and on microsomal protein(s). We posited that membrane topology of the amino-terminal third of the H(+)-ATPase, and perhaps of other P-type ATPases is achieved by inserting transmembrane segments into membrane in pairs.
Amino Acid Sequence↗
The membrane topology of the carboxyl-terminal third of the Neurospora plasma membrane H(+)-ATPase.
To localize transmembrane segments in the carboxyl-terminal third of the Neurospora plasma membrane H(+)-ATPase, we constructed fusion proteins on the cDNA level. These contained DNA fragments encoding hydrophilic residues of the amino and carboxyl termini of the H(+)-ATPase with a DNA fragment encoding the putative transmembrane segment. To report translocation into microsomes, a DNA fragment encoding three consensus N-linked glycosylation sites was engineered carboxyl-terminal to the putative transmembrane segment. Fusion proteins were synthesized in a Neurospora in vitro translation system supplemented with homologous microsomes. By the criteria of glycosylation of fusion proteins by microsomes, sedimentation of products with microsomes after alkaline extraction, and analysis of protected fragments generated from proteinase K digestion of integrated products, we localized six transmembrane segments in the carboxyl-terminal third of the H(+)-ATPase. These results support a 10-segment model of the Neurospora H(+)-ATPase.
Amino Acid Sequence↗
8-Azido-ATP modification of cytochrome c: retardation of its electron-transfer activity to cytochrome c oxidase.
Horse heart cytochrome c has been modified by 8-azido-ATP and the electron-transfer activity of the modified cytochrome c's to bovine heart cytochrome c oxidase (CcO) under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The intermolecular electron transfer between the redox protein partners was shown to be extremely slow. The 8-azido-ATP-modified system exhibited less than 5% of the intracomplex electron-transfer rate observed between native cytochrome c and CcO under otherwise identical conditions. The binding affinity of the modified cytochrome c was greatly reduced (3 orders of magnitude) at low ionic strengths; however, it was only slightly reduced (by a factor of 2) relative to the native protein at physiological ionic strengths. Thus, the binding affinity of the ATP-cytochrome c adducts is relatively insensitive to the ionic strength compared to the native enzyme, suggesting that a different docking conformation is assumed by the ATP-cytochrome c adducts in their interaction with the oxidase. Since the redox potential of the modified cytochrome c is close to the value of its native form, we conclude that there has been a change in the docking of the cytochrome c to CcO and the electronic coupling between heme c and CuA upon 8-azido-ATP modification.
Adenosine Triphosphate↗
Sarcoma botryoides of the cervix treated with limited surgery and chemotherapy to preserve fertility.
Sarcoma botryoides rarely arises in the uterine cervix. It usually presents in the adolescent age group and is treated primarily by surgery followed by adjuvant chemotherapy. It has been reported in the literature that is possible to limit surgery to local excision in stage I cases, when the tumor is confined to the cervix. In an attempt to preserve reproductive function in a young female patient, we performed cervicectomy alone, followed by adjuvant chemotherapy. After 36 months follow-up she remains well, suggesting that limited excision with adjuvant chemotherapy may be sufficient for early-stage disease.
Adult↗
Effect of platelet-activating factor on secretion of mucous glycoprotein from chinchilla middle ear epithelial cells in vitro.
Platelet-activating factor (PAF) is a naturally occurring phospholipid that acts as a pleiotropic mediator and mediates cell-cell reactions under physiological and pathological conditions. Recently, it has been shown that PAF is a strong secretagogue of mucous glycoprotein in the airways, suggesting its role in mucous glycoprotein secretion and the pathogenesis of otitis media with effusion. In the current study, we examined the effect of PAF on mucous glycoprotein secretion in cultured chinchilla middle ear epithelial cells. PAF at 1 microM significantly stimulated mucous glycoprotein secretion from cultured chinchilla middle ear epithelial cells. This action was concentration-dependent, with secretions reaching near maximum when the cells were incubated with PAF at 100 microM. In a time-dependent study, PAF demonstrated an initial rapid stimulation of mucous glycoprotein secretion, followed by a gradual increase thereafter. A six-fold increase was seen in the first 2 h compared with controls. Cycloheximide, a protein synthesis inhibitor, demonstrated an inhibitory effect on PAF-stimulated mucous glycoprotein secretion in this study. These findings suggest that PAF plays an important role in the pathogenesis of otitis media with effusion by stimulating mucous glycoprotein secretion in vitro.
Animals↗