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Biomedical subjects

R H Chen

Publications and source records attributed to R H Chen.

At least 91 records · Page 5Linked to original sources
PubMed1993

Inactivation of the type II receptor reveals two receptor pathways for the diverse TGF-beta activities.

Transforming growth factor-beta (TGF-beta) is a multifunctional protein that regulates cell proliferation and differentiation and extracellular matrix production. Although two receptor types, the type I and type II receptors, have been implicated in TGF-beta-induced signaling, it is unclear how the many activities of TGF-beta are mediated through these receptors. With the use of cells overexpressing truncated type II receptors as dominant negative mutants to selectively block type II receptor signaling, the existence of two receptor pathways was shown. The type II receptors, possibly in conjunction with type I receptors, mediate the induction of growth inhibition and hypophosphorylation of the retinoblastoma gene product pRB. The type I receptors are responsible for effects on extracellular matrix, such as the induction of fibronectin and plasminogen activator inhibitor I, and for increased JunB expression. Selective inactivation of the type II receptors alters the TGF-beta response in a similar manner to the functional inactivation of pRB, suggesting a role for pRB in the type II, but not the type I, receptor pathway.

Animals↗
PubMed1993

Cloning of a type I TGF-beta receptor and its effect on TGF-beta binding to the type II receptor.

Transforming growth factor-beta (TGF-beta) affects cellular proliferation, differentiation, and interaction with the extracellular matrix primarily through interaction with the type I and type II TGF-beta receptors. The type II receptors for TGF-beta and activin contain putative serine-threonine kinase domains. A murine serine-threonine kinase receptor, Tsk 7L, was cloned that shared a conserved extracellular domain with the type II TGF-beta receptor. Overexpression of Tsk 7L alone did not increase cell surface binding of TGF-beta, but coexpression with the type II TGF-beta receptor caused TGF-beta to bind to Tsk 7L, which had the size of the type I TGF-beta receptor. Overexpression of Tsk 7L inhibited binding of TGF-beta to the type II receptor in a dominant negative fashion. Combinatorial interactions and stoichiometric ratios between the type I and II receptors may therefore determine the extent of TGF-beta binding and the resulting biological activities.

Amino Acid Sequence↗
PubMed1993

Activation of MAP kinases, pp90rsk and pp70-S6 kinases in mouse mast cells by signaling through the c-kit receptor tyrosine kinase or Fc epsilon RI: rapamycin inhibits activation of pp70-S6 kinase and proliferation in mouse mast cells.

The high-affinity receptor for IgE, Fc epsilon RI, represents the major cell surface structure through which mast cells express immunologically specific secretory function. By contrast, the stem cell factor receptor (SCFR), which is encoded by c-kit, is essential for normal mast cell development. The signaling pathways initiated by the stimulation of mast cells through the Fc epsilon RI, which lacks intrinsic kinase activity, and the SCFR, a member of the receptor tyrosine kinase family, generally have been regarded to be distinct. We report here that mouse mast cells stimulated either with SCF or with IgE and specific antigen exhibit a remarkably similar pattern of activation of mitogen-activated protein kinases (MAPK), 90 kDa-S6 kinases (pp90rsk), and pp70-S6 kinases (pp70-S6K). These results indicate that all three families of protein kinases are associated with the cell surface receptor-dependent activation of secretion, as well as proliferation, in mast cells. We also show that the immunosuppressant rapamycin, but not FK506, can inhibit both SCF-dependent pp70-S6 kinase activation and SCF-dependent proliferation in mouse mast cells, without suppressing IgE- and antigen-dependent mediator release. These findings suggest that the activation of pp70-S6 kinase represents an important link in the stimulation of cell proliferation by SCF. Our results also indicate that the intracellular signaling pathways initiated by stimulation of mast cells through the Fc epsilon RI or the SCFR exhibit more overlap than has previously been appreciated.

Animals↗
PubMed1993

The use of granzyme A as a marker of heart transplant rejection in cyclosporine or anti-CD4 monoclonal antibody-treated rats.

Granzyme A is a serine protease expressed by populations of human and mouse natural killer cells and activated CD4+ and CD8+ cytotoxic lymphocytes; its expression marks a subset of inflammatory cells in allografts, autoimmune diabetes, and a number of other inflammatory lesions. In order to describe more completely the correlation between granzyme A expression and the presence of in vivo cytolytic effects, we grafted allogeneic rat hearts with vascular anastomoses in a heterotopic location, and treated the hosts with either cyclosporine, anti-CD4 monoclonal antibody (MRC OX38), or no therapy. The grafts were evaluated by palpation for cardiac functions, by immunohistochemistry for CD4/CD8 expression, by hematoxylin-and-eosin staining for inflammatory infiltration, and by in situ hybridization for granzyme A expression. The appearance of granzyme A+ cells in untreated allografts preceded both functional and standard histopathological and immunohistochemical evidence of graft rejection by two days. In donor-recipient combinations where cyclosporine and anti-CD4 treatments allowed indefinite allograft survival, the allografts showed minimal numbers of granzyme A+ cells, whether cellular infiltrates developed or not. The number of granzyme A+ cells present in the cardiac allografts in treated and untreated animals correlated with either current or impending episodes of rejection. The early time course of granzyme A expression suggests that it can be used as an early and reliable marker of graft rejection.

Animals↗
PubMed1993

A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity.

A signaling pathway by which growth factors may induce transcription of the c-fos proto-oncogene has been characterized. Growth factor stimulation of quiescent fibroblasts activates a protein kinase cascade that leads to the rapid and transient phosphorylation of the serum response factor (SRF), a regulator of c-fos transcription. The in vivo kinetics of SRF phosphorylation and dephosphorylation parallel the activation and subsequent repression of c-fos transcription, suggesting that this phosphorylation event plays a critical role in the control of c-fos expression. The ribosomal S6 kinase pp90rsk, a growth factor-inducible kinase, phosphorylates SRF in vitro at serine 103, the site that becomes newly phosphorylated upon growth factor stimulation in vivo. Phosphorylation of serine 103 significantly enhances the affinity and rate with which SRF associates with its binding site, the serum response element, within the c-fos promoter. These results suggest a model in which the growth factor-induced phosphorylation of SRF at serine 103 contributes to the activation of c-fos transcription by facilitating the formation of an active transcription complex at the serum response element.

3T3 Cells↗
PubMed1993

Differential transcriptional activation by v-myb and c-myb in animal cells and Saccharomyces cerevisiae.

The v-myb oncogene and its cellular homolog c-myb encode sequence-specific DNA-binding proteins which regulate transcription from promoters containing Myb-binding sites in animal cells. We have developed a Saccharomyces cerevisiae system to assay transcriptional activation by v-Myb and c-Myb. In yeast strains containing integrated reporter genes, activation was strictly dependent upon both the Myb DNA-binding domain and the Myb recognition element. BAS1, an endogenous Myb-related yeast protein, was not required for transactivation by animal Myb proteins and by itself had no detectable effect on a Myb reporter gene. Deletion analyses demonstrated that a domain of v-Myb C terminal to the previously mapped Myb transcriptional activation domain was required for transactivation in animal cells but not in S. cerevisiae. The same domain is also required for the efficient transformation of myeloid cells by v-Myb. In contrast to results in animal cells, in S. cerevisiae the full-length c-Myb was a much stronger transactivator than a protein bearing the oncogenic N- and C-terminal truncations of v-Myb. These results imply that negative regulation of c-Myb by its own termini requires an additional animal cell protein or small molecule that is not present in S. cerevisiae.

Amino Acid Sequence↗
PubMed1993

Functional domains and phosphorylation of the orphan receptor Nur77.

Nur77 represents a unique class within the steroid receptor superfamily since its synthesis is tightly regulated by extracellular signals and it is capable of potent transactivation activity in the absence of an exogenously added ligand. In this study, we sought to dissect the functional domains regulating the activities of Nur77 by deletion mapping. We demonstrate that whereas the transactivation activity of Nur77 resides in the amino-terminal domain, the carboxy-terminal domain regulates this activity. A short deletion from the carboxy terminus eliminates transactivation activity while a further deletion restores the activity. Deletion of the domain immediately carboxyl to the zinc fingers motif eliminates both DNA binding activity and nuclear localization, thus abolishing transactivation. Nur77 is posttranslationally modified predominantly by phosphorylation, which occurs primarily at the N-terminal domain. The growth-related kinase pp90rsk, but neither the pp44mapk nor the pp70s6k, can phosphorylate recombinant Nur77 in vitro. Furthermore, we have identified a site within the region required for sequence-specific DNA binding, Ser-354, that is phosphorylated by pp90rsk in vitro; this site is also phosphorylated in vivo. The possibility that phosphorylation might affect DNA binding is discussed.

3T3 Cells↗
PubMed1993

Calbistrins, novel antifungal agents produced by Penicillium restrictum. II. Isolation and elucidation of structure.

The novel antifungal agents, calbistrins A, B, C and D have been isolated from a strain of Penicillium restrictum (AB 1875C-28). The four congeners were separated by bioactivity directed fractionation using countercurrent chromatography and preparative-HPLC. NMR studies revealed that the calbistrins each contain a carboxylic acid conjugated tetraene attached through an aliphatic ester linkage to a hexahydronaphthalene system.

Antifungal Agents↗
PubMed1993

[The diagnosis of pleural diseases with a fiberbronchoscope under open pneumothorax].

Thoracoscopy, using a fiberbronchoscope under open pneumothorax, was performed in 27 cases suffering from pleural diseases undiagnosed. The overall diagnostic rate was 92.3%. The items observed included pulse rate, respiration rate, blood pressure, blood gas analysis, electrocardiography and thoracic pressure measure. It was found that this method has little influence on cardiopneumatic function, no harm fiberbronchoscope, and is simple and safe.

Adult↗
PubMed1993

In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression.

We report a novel method for detecting intracellular messenger RNA by combining the techniques of in situ hybridization and polymerase chain reaction (PCR) (in situ cDNA PCR). The technique could detect low abundancy signals and distinguish different levels of gene expression. We examined the expression of the functional markers of activated cytotoxic T lymphocytes, granzyme A, and perforin in human lymphocytes from in vitro cultures. The amplification products were found in the cells and the supernatants, with the distribution critically affected by the protease digestion conditions. The specificity of amplification was confirmed by electrophoretic analysis and Southern blotting. We conclude that the in situ cDNA PCR technique offers a sensitive method of measuring the frequency of signal-expressing cells and has significant research and clinical applications.

Biopsy↗
PubMed1992

Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in the HPRT gene of diploid human fibroblasts.

If excision repair-proficient human cells are allowed time for repair before onset of S phase, the premutagenic lesions formed by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, BPDE) are lost from the transcribed strand of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene faster than from the nontranscribed strand. No change in strand distribution is seen with repair-deficient cells. These results suggest strand-specific repair of BPDE-induced DNA damage in human cells. To test this, we measured the initial number of BPDE adducts formed in each strand of the actively transcribed HPRT gene and the rate of repair, using UvrABC excinuclease in conjunction with Southern hybridization and strand-specific probes. We also measured the rate of loss of BPDE adducts from the inactive 754 locus. The frequencies of adducts formed by exposure to BPDE (1.0 or 1.2 microM) in either strand of a 20-kilobase fragment that lies entirely within the transcription unit of the HPRT gene were similar; the frequency in the 14-kilobase 754 fragment was approximately 20% lower. The rates of repair in the two strands of the HPRT fragment differed significantly. Within 7 hr after treatment with 1.2 microM BPDE, 53% of the adducts had been removed from the transcribed strand, but only 26% from the nontranscribed strand; after 20 hr, these values were 87% and 58%, respectively. In contrast, only approximately 14% of the BPDE adducts were lost from the 754 locus in 20 hr, a value even lower than the rate of loss from the overall genome (i.e., 38%). These results demonstrate strand-specific and preferential repair of BPDE adducts in human cells. They suggest that the heterogeneous repair of BPDE adducts in the human genome cannot be accounted for merely by the greatly increased rate of the repair specific to the transcribed strand of the active genes, and they point to a role for the chromatin structure.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1992

Nuclear localization and regulation of erk- and rsk-encoded protein kinases.

We demonstrate that members of the erk-encoded family of mitogen-activated protein (MAP) kinases (pp44/42mapk/erk) and members of the rsk-encoded protein kinases (RSKs or pp90rsk) are present in the cytoplasm and nucleus of HeLa cells. Addition of growth factors to serum-deprived cells results in increased tyrosine and threonine phosphorylation and in the activation of cytosolic and nuclear MAP kinases. Activated MAP kinases then phosphorylate (serine/threonine) and activate RSKs. Concurrently, a fraction of the activated MAP kinases and RSKs enter the nucleus. In addition, a distinct growth-regulated RSK-kinase activity (an enzyme[s] that phosphorylates recombinant RSK in vitro and that may be another member of the erk-encoded family of MAP kinases) was found associated with a postnuclear membrane fraction. Regulation of nuclear MAP kinase and RSK activities by growth factors and phorbol ester is coordinate with immediate-early gene expression. Indeed, in vitro, MAP kinase and/or RSK phosphorylates histone H3 and the recombinant c-Fos and c-Jun polypeptides, transcription factors phosphorylated in a variety of cells in response to growth stimuli. These in vitro studies raise the possibility that the MAP kinase/RSK signal transduction pathway represents a protein-Tyr/Ser/Thr phosphorylation cascade with the spatial distribution and temporal regulation that can account for the rapid transmission of growth-regulating information from the membrane, through the cytoplasm, and to the nucleus.

Amino Acid Sequence↗
PubMed1991

Lack of a cell cycle-dependent strand bias for mutations induced in the HPRT gene by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in excision repair-deficient human cells.

We showed previously that in repair-proficient human cells the location of the premutagenic lesion induced by (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo(a)pyrene (BPDE), namely, the guanine in a G.C base substitution, in mutants derived from cells treated at the beginning of S phase just when the hypoxanthine (guanine) phosphoribosyltransferase gene is replicated, differs significantly from their location in cells treated 12 h prior to the beginning of S phase (early G1 phase) (R-H. Chen et al., Proc. Natl. Acad. Sci. USA, 87:8680-8684, 1990). This suggests that the cells preferentially remove BPDE adducts from the transcribed strand. We have now determined the kinds and location of independent mutations induced by BPDE in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene of synchronized repair-deficient xeroderma pigmentosum cells (XP12BE, complementation group A), treated at S or in G1. Nineteen of 25 mutants derived from S-treated cells and 23 of 28 mutants from G1-treated cells contained base substitutions. Eighty-nine percent of these involved a G.C base pair, primarily G.C----T.A transversions. This is similar to the kinds of mutations we saw in the repair-proficient cells. However, in contrast to our earlier results, there was no change in strand distribution of premutagenic BPDE lesions. In both populations, approximately 26% of the base substitutions involving G.C base pairs had the G located in the transcribed strand, 5 of 18 in the S phase mutants, and 5 of 21 in the G1 phase mutants. These results support the hypothesis that the strong strand bias of induced mutations observed in the repair-proficient cells results from preferential repair of BPDE-induced DNA damage from the transcribed strand.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Use of PCR amplification of cDNA to study mechanisms of human cell mutagenesis and malignant transformation.

PCR is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. But some investigators need to sequence the entire coding region of mammalian genes to determine what specific changes have occurred. In 1989, we [Yang et al: Gene 83:347-354] described a method to copy mRNA of the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene directly from the lysate of a clone of 6-thioguanine-resistant mutant diploid human fibroblasts without the need for RNA extraction or DNA template purification. To avoid detecting random changes introduced by polymerases, 100 to 500 cells from an individual clone, each containing the identical mutation, are lysed and the cDNA is amplified 10(10)-to 10(11)-fold to obtain 5 to 10 micrograms of DNA. The consensus sequence of the cDNA is determined by direct nucleotide sequencing. Using this method, we have investigated the kinds of mutations induced by carcinogens in the coding region of the HPRT gene and their location in the gene and examined the role of DNA repair in this process. Normal repair-proficient human cells and cells deficient in DNA repair were exposed to mutagens in exponential growth or synchronized and exposed at the beginning of S phase or in G1 phase several hr prior to DNA replication. The kinds and location of mutations in the HPRT gene were determined and knowledge of the nature of the DNA lesions formed by the various mutagens allowed assignment of the DNA strand in which the premutagenic lesion that gave rise to the mutation had been located. Related assays involving PCR have been used to determine the nature of mutations in the coding region of the H-, N-, or K-ras genes of tumor-derived malignant human cells and to determine whether or not such cells express specific growth factor genes.

Cell Transformation, Neoplastic↗
PubMed1991

Kinds and location of mutations induced by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in diploid human fibroblasts.

To gain insight into the mechanisms by which mutations are induced in human cells by carcinogens, we have determined the kinds and location (spectrum) of mutations induced in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene by (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Individual populations of diploid human fibroblasts were treated with BPDE, or were left untreated (control). After a suitable expression period, the progeny cells were selected for resistance to 6-thioguanine. Individual drug-resistant colonies were isolated, and the mRNA in the lysate of 100-400 cells from each colony was copied directly into cDNA using reverse transcriptase. The cDNA of the HPRT gene of 29 unequivocally independent mutants from BPDE-treated populations and 13 from the control populations was amplified 10(11)-fold, and the product was sequenced directly. Twenty-three of the 29 BPDE-induced mutants examined contained a single base pair substitution; four exhibited two base pair substitutions. Eight out of 13 control mutants exhibited base pair substitutions, and four others were missing a complete exon. Thirty of the 32 base pair substitutions in the BPDE-induced mutants involved G.C base pairs, primarily G.C----T.A transversions. The majority (89%) of the base pair substitutions observed in the mutants from the control population involved an A.T base pair. Base substitutions were found throughout the coding region of the gene, but 41% of those seen in mutants from the BPDE-treated population and 44% of those from the untreated population were located in the first half of exon 3.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Regulation of pp90rsk phosphorylation and S6 phosphotransferase activity in Swiss 3T3 cells by growth factor-, phorbol ester-, and cyclic AMP-mediated signal transduction.

Somatic cell homologs to the Xenopus laevis S6 protein kinases (referred to collectively as pp90rsk) have recently been identified and partially characterized. Here we examine alterations in pp90rsk phosphorylation and S6 phosphotransferase activity in response to regulators of multiple signal transduction systems: purified growth factors, phorbol ester, changes in cyclic AMP (cAMP) levels, and sodium vanadate. All reagents tested increased pp90rsk serine and threonine phosphorylation, but only those agents that regulate cell proliferation and sodium vanadate activated its S6 kinase activity. In addition to the cAMP-stimulated phosphorylation of pp90rsk, a simple correlation between the extent of growth-regulated pp90rsk phosphorylation and S6 phosphotransferase activity was not observed. Quantitative phosphorylation of pp90rsk continued to increase after its S6 kinase activity began its return towards basal levels. However, a close correlation between the appearance and disappearance of a slow-mobility form of phosphorylated pp90rsk (by electrophoresis) and pp90rsk activity was observed. In addition, pp90rsk was regulated by both protein kinase C-independent and -dependent signaling mechanisms. The extent of protein kinase C participation, however, varied depending on which growth factor receptor was activated. Furthermore, growth factor-specific differences in the temporal regulation of pp90rsk S6 phosphotransferase activity were also observed. These results support the notion that the complex regulation of the rsk gene product constitutes one of the primary responses of animal cells to mitogenic signals.

Animals↗