Biomedical subjects

E S Hwang

Publications and source records attributed to E S Hwang.

At least 37 records · Page 2Linked to original sources

PubMed1998

The regulation of Cdc20 proteolysis reveals a role for APC components Cdc23 and Cdc27 during S phase and early mitosis.

BACKGROUND: In eukaryotic cells, a specialized proteolysis machinery that targets proteins containing destruction-box sequences for degradation and that uses a ubiquitin ligase known as the anaphase-promoting complex/cyclosome (APC) plays a key role in the regulation of mitosis. APC-dependent proteolysis triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Recently, two highly conserved WD40-repeat proteins, Cdc20 and Cdh1/Hct1, have been identified as substrate-specific regulators for APC-dependent proteolysis in the budding yeast Saccharomyces cerevisiae. Here, we have investigated the cell cycle regulation of Cdc20 and Cdh1/Hct1. RESULTS: Whereas the levels CDH1/HCT1 RNA and Cdh1/Hct1 protein are constant throughout the cell cycle, CDC20 RNA and Cdc20 protein are present only during late S phase and mitosis and Cdc20 protein is unstable throughout the entire cell cycle. The instability of Cdc20 depends on CDC23 and CDC27, which encode components of the APC. During the G1 phase, a destruction box within Cdc20 mediates its instability, but during S phase and mitosis, although Cdc20 destruction is still dependent on CDC23 and CDC27, it does not depend on the Cdc20 destruction box. CONCLUSIONS: There are remarkable differences in the regulation of Cdc20 and Cdh1/Hct1. Furthermore, the APC activator Cdc20 is itself a substrate of the Cdc27 have a role in the degradation of Cdc20 during S Phase and early mitosis that is not mediated by its destruction box.

Anaphase-Promoting Complex-Cyclosome↗

PubMed1998

Novel CRE-binding proteins of 11-16 kDa bind to the LDH A-gene CRE in a sequence specific and hepatocyte-growth dependent manner in partially hepatectomized rat liver.

We examined cAMP response element (CRE)-binding proteins involved in lactate dehydrogenase A (LDHA)-gene transcription in rat liver after partial hepatectomy. Gel retardation and Southwestern blot assays showed that the CRE-binding activity of the 11-16 kDa novel proteins increased in accordance with increases in LDH A-mRNA in regenerating liver tissues, whereas that of the 43 kDa CREB did not. Using CRE-oligonucleotide affinity chromatography and reverse-phase HPLC, we purified four CRE-binding proteins of 11.2, 15.2, 15.8, and 16.3 kDa. N-terminal amino acid sequences of 15.2 and 16.3 kDa proteins revealed a high sequence homology to but were not identical with those of rat histone H2A.1 and H2B, respectively. CRE-bindings of these two proteins were highly specific, while those of histones H2A.1 and H2B were nonspecific as shown by competition-Southwestern blot and DNase I footprinting assays. Taking these data together, we suggest that the novel 11-16 kDa CRE-binding proteins are responsible for the cell growth-dependent inducibility of LDH A-gene transcription during liver regeneration.

Amino Acid Sequence↗

PubMed1998

Budding yeast Cdc20: a target of the spindle checkpoint.

The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.

Amino Acid Sequence↗

PubMed1998

Volume of resection in patients treated with breast conservation for ductal carcinoma in situ.

BACKGROUND: The optimal treatment of ductal carcinoma in situ (DCIS) is one of the most controversial issues in the management of breast cancer. Identification of factors that affect the risk of local recurrence is very important as the incidence of DCIS increases and the use of breast conservation becomes more widespread. Because the extent of resection may affect the relapse rate, we hypothesized that larger volumes of resection (VR) may account for the lower local recurrence rates we have previously found in elderly patients. METHODS: Between 1978 and 1990, 173 cases of histologically confirmed DCIS were treated at MSKCC with breast conservation therapy. Of these, complete VR data were available for 126 cases. The VRs thus obtained were divided into two groups, <60 cm3 and > or =60 cm3, and were evaluated for correlating factors. The patients were divided into three groups by age at diagnosis: younger than 40 years, 40 to 69 years, and 70 years or older. RESULTS: The eldest group had a significantly greater proportion of large VRs (30%) as compared to the middle group (11%) and the youngest group (9%) (P=.03, chi2). Although not statistically significant, the large VR group had a lower 6-year actuarial local recurrence rate (5.6%) than did the small VR group (21.3%) (P=.16, log-rank test). This trend was observed even though adjuvant radiotherapy was used less often in patients who had large VRs. CONCLUSION: Breast conservation surgery for DCIS in elderly patients is more likely to employ a large VR. This may explain, at least in part, the observation that elderly patients have a lower local recurrence rate.

Adult↗

PubMed1998

The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation.

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs) by an unknown mechanism. We show that the Cdc14 phosphatase triggers mitotic exit by three parallel mechanisms, each of which inhibits Cdk activity. Cdc14 dephosphorylates Sic1, a Cdk inhibitor, and Swi5, a transcription factor for SIC1, and induces degradation of mitotic cyclins, likely by dephosphorylating the activator of mitotic cyclin degradation, Cdh1/Hct1. Feedback between these pathways may lead to precipitous collapse of mitotic CDK activity and help coordinate exit from mitosis.

Anaphase-Promoting Complex-Cyclosome↗

PubMed1998

Does the proven benefit of mammography extend to breast cancer patients over age 70?

BACKGROUND: Prospective randomized studies show reduced breast cancer mortality among women offered mammographic screening; yet, few women 70 or older were represented in these trials. We examine the impact of mammography on stage at diagnosis of breast cancer, over the years when mammography came into general use, comparing women aged 40 to 69 with those aged 70 and older. METHODS: We reviewed the records of 1,001 consecutive patients 40 and older treated for invasive or in situ breast cancer in the surgical practice of one of us (H.S.C.) between 1979 and 1993, comparing trends in mammography use, means of diagnosis, tumor size, axillary node status, and pathology. RESULTS: The proportion of cases diagnosed by mammography increased over time to a comparable degree in both age groups, as did the proportion of T1 and DCIS or microinvasive cancers. This trend toward earlier stage appears entirely due to an increasing use of mammography. CONCLUSION: The potential benefit of regular mammography to healthy women aged 70 and older may equal that observed in their younger counterparts.

Age Factors↗

PubMed1997

Co-expression of urokinase-type plasminogen activator and its receptor in human gastric-cancer cell lines correlates with their invasiveness and tumorigenicity.

The expression of urokinase-type plasminogen activator (u-PA), its receptor (u-PAR) and metalloproteases activity were analyzed in 4 human gastric-cancer cell lines (AGS, Hs746T, SNU-1, and SNU-5), in an attempt to relate these activities to their invasive potential and tumorigenicity on the modified chorioallantoic membranes (CAM) of chick embryos. Only 1 of the 4 cell lines tested, Hs746T, expressed both u-PA and u-PAR as well as MMP-2, but not MMP-9. This cell line was both tumorigenic and highly invasive (51.3 +/- 13.1%) on a modified CAM. Its invasive capacity was comparable with that of a highly malignant human epidermoid-carcinoma cell line (HEp3), which usually showed 40 to 50% invasiveness. The 3 other cell lines all produced MMP-2 and MMP-9, but only AGS showed moderate invasiveness (24.2 +/- 8.8%). While antibodies to u-PA were significantly effective in reducing CAM invasiveness of Hs746T cells by approximately 40%, the invasiveness of the t-PA-expressing AGS cell line was not affected by anti-t-PA antibodies. These results suggest that when one of the components of the u-PA/u-PAR system (the enzyme and/or the receptor) is not produced and u-PA/u-PAR-dependent cell-surface proteolytic activity is thereby diminished, the malignant phenotype that can be determined by tumorigenicity and invasion of connective tissue on a CAM is compromised. Production of both type-IV collagenases (MMP-2 and MMP-9) cannot offset this deficiency.

Animals↗

PubMed1997

Type II protein kinase A up-regulation is sufficient to induce growth inhibition in SK-N-SH human neuroblastoma cells.

We have previously reported that overexpression of RII beta subunit of protein kinase A, which markedly reduces RI alpha protein, induces growth inhibition in SK-N-SH human neuroblastoma cells. To determine whether the reduction of RI alpha or protein kinase A isozyme type I is essential in the growth inhibition of SK-N-SH cells, we overexpressed RI alpha in sense and antisense orientation. Type I protein kinase A activity was increased in the RI alpha-overexpressing cells and was decreased in the RI alpha antisense-expressing cells. However, the changes in type I protein kinase A activities did not affect cell growth. Overexpression of RII beta or C alpha increased type II protein kinase A and inhibited cell growth in both cell lines regardless of the type I protein kinase A level. These results indicate that type II protein kinase A is the main effector in the cAMP-mediated growth regulation of SK-N-SH human neuroblastoma cells.

Affinity Labels↗

PubMed1997

Physical status and expression of HPV genes in cervical cancers.

It is known that E2 protein of oncogenic human papillomavirus (HPV) inhibits the expression of E6 and E7 genes from their major promoters in vitro and suppresses the proliferation of cervical cancer cells. This suggests that the loss of functional E2 gene may provide selective advantages in the development of cervical cancer. Investigation of the relationship between the disruption of HPV-16/18 E2 genes by DNA integration and clinical outcome of cervical cancer may not only help to understand the mechanism of HPV-related cervical carcinogenesis, but may also provide novel management of cervical cancer. It was noted that integrated HPV-16/18 DNA was predominant in most patients with cervical cancers, marking 51 of a total of 68 cases (75%); episomal HPV DNAs were found in 5 cases (7.4%), and finally mixed forms of HPV DNAs with episome and integration were found in 12 cases (17.6%). Whole portions of E2 DNA of HPV-16 could be amplified by PCR in 19 (36.5%) of 52 cases of cervical intraepithelial neoplasia. It was shown that there was not statistically significant association with the different stages, but integrated HPV DNAs were detectable only in the patients with far-advanced stage of cervical cancers, which also means no episomal forms were detected. Episomal forms of HPV DNA were detectable in 14 (25.9%) of 54 squamous cell carcinomas (4 pure episomal forms and 10 mixed forms), whereas only 1 (8.3%) of 12 adenocarcinomas and adenosquamous cell carcinomas contained episomal viral DNA. When HPV DNA forms were compared with initial tumor size, lymphovascular space involvement, and frequency of nodal metastasis, statistically significant relationships were not found. The association of DNA integration with invasive cervical cancers was seen regardless of HPV type; however, there were differences between the integration profiles of HPV-16 and HPV-18 DNA. Of the 51 HPV-16-containing cancers, 36 (70.6%) revealed purely integrated HPV DNA, and another 10 cases (19.6%) displayed both integrated and episomal HPV DNAs. However, 5 (9.8%) cases showed only episomal copies of the HPV-16 genome. In contrast, all 17 HPV-18-containing cancers (5 cases positive for HPV-18 and 12 cases positive for both HPV-16 and -18) revealed only the integrated form of HPV-18 DNA. The expression of E6 and E7 transcripts of HPV-16/18 is uniformly correlated with the physical status of HPV DNAs. HPV E2 mRNAs were constantly expressed in the presence of the intact virus in cases with episome and mixed forms of HPV DNA. In general, amplified signals from HPV E2 RT-PCR are more intensive than those from DNA-PCR in the same patients. It is suggested that RT-PCR is a valuable method to evaluate dynamic expression of the specific gene and seems to be more sensitive than the DNA-PCR method in detecting intact E2 gene because of the gene copy numbers.

Base Sequence↗

PubMed1997

dl-propranolol negatively regulates the transcription of proliferating cell nuclear antigen (PCNA)-gene and thereby suppresses DNA synthesis in regenerating rat liver.

Previous reports have suggested that dl-propranolol (PRL) suppresses DNA synthesis by blocking cAMP-mediated signaling in rat liver after partial hepatectomy (PH). Here, we examined if PRL negatively regulates the expression of genes involved in cell cycle progression. Immunoblotting assays showed that the protein levels of cyclins A and E, Cdk2, p21WAF1, and p27KIP1 did not significantly change in liver tissues from either vehicle- or PRL-injected rats after PH. However, the levels of PCNA and PCNA-mRNA markedly decreased in the remnant liver in response to PRL-injection. Similarly, PCNA-CRE binding activity of nuclear 43kDa CREB was suppressed, although the protein levels were not altered. We suggest that PRL negatively regulates the PCNA-gene transcription by interfering with the cAMP/PKA-mediated induction of CREB binding to the CRE-sequences and thereby suppresses DNA synthesis in regenerating rat liver.

Animals↗

PubMed1997

The human cytomegalovirus UL55 (gB) and UL75 (gH) glycoprotein ligands initiate the rapid activation of Sp1 and NF-kappaB during infection.

The cellular transcription factors Sp1 and NF-kappaB were upregulated shortly after the binding of purified live or UV-inactivated human cytomegalovirus (HCMV) to the cell surface. The rapid time frame of transcription factor induction is similar to that seen in other systems in which cellular factors are induced following receptor-ligand engagement. This similarity suggested that a cellular receptor-viral ligand interaction might be involved in Sp1 and NF-kappaB activation during the earliest stages of HCMV infection. To focus on the possible role viral ligands play in initiating cellular events following infection, we first used purified viral membrane extracts to demonstrate that constituents on the membrane are responsible for cellular activation. Additionally, these studies showed, through the use of neutralizing antibodies, that the viral membrane mediators of this activation are the major envelope glycoproteins gB (UL55) and gH (UL75). To confirm these results, neutralizing anti-gB and -gH antibodies were used to block the interactions of these glycoproteins on whole purified virus with their cell surface receptors. In so doing, we found that Sp1 and NF-kappaB induction was inhibited. Lastly, through the use of purified viral gB protein and an anti-idiotypic antibody that mimics the image of the viral gH protein, it was found that the engagement of individual viral ligands with their appropriate cell surface receptors was sufficient to activate cellular Sp1 and NF-kappaB. These results support our hypothesis that HCMV glycoproteins mediate an initial signal transduction pathway which leads to the upregulation of host cell transcription factors and suggests a model wherein the orderly sequence of virus-mediated changes in cellular activation initiates with viral binding via envelope glycoproteins to the cognate cellular receptor(s).

Antibodies, Anti-Idiotypic↗

PubMed1996

Activation of the endogenous p53 growth inhibitory pathway in HeLa cervical carcinoma cells by expression of the bovine papillomavirus E2 gene.

We previously showed that expression of the bovine papillomavirus (BPV) E2 gene results in a dramatic inhibition of the proliferation of several human cervical carcinoma cell lines, including HeLa cells which contain human papillomavirus (HPV) type 18 DNA. We have assessed the status of endogenous G1 cell cycle regulatory proteins, including the tumor suppressor proteins, p53 and p105Rb, in order to investigate growth regulatory pathways in HeLa cells following E2 expression. The p53 tumor suppressor protein is stabilized following the introduction of the E2 gene into HeLa cells. This results in the induction of the p53-responsive gene encoding the cyclin dependent kinase (cdk) inhibitor, p21/WAF1, complex formation between p21/WAF1 and cdk2 and reduction of in vitro cdk2/cyclin E kinase activity. The reduced cdk kinase activity is accompanied by the accumulation of the growth inhibitory hypophosphorylated form of the tumor suppressor protein, p105Rb. The level of the p105Rb-regulated transcription factor, E2F1, is reduced, as is transcription of a variety of E2F1-regulated genes, including B-myb. Thus, the p53 growth inhibitory pathway has evidently not accumulated mutations in HeLa cells but rather appears intact. However, this pathway remains dormant, until it is mobilized by appropriate manipulations, such as the expression of the BPV E2 protein.

Bovine papillomavirus 1↗

PubMed1996

Identification of a variant estrogen receptor lacking exon 4 and its coexpression with wild-type estrogen receptor in ovarian carcinomas.

By means of reverse transcription-PCR we have identified an alternatively spliced mRNA coding for a variant estrogen receptor (ER) that lacks exon 4 (ERDelta4) and is coexpressed with the wild-type ER mRNA in ovarian carcinomas. Furthermore, Western blot analysis revealed the expression of the ERDelta4 protein in normal as well as neoplastic ovarian tissues along with the wild-type ER, although the relative amounts of the wild-type ER and ERDelta4 proteins varied. The trans-activational properties of this variant were studied in ER-negative COS1 cell lines by cotransfection of the ERDelta4 expression vector and a reporter gene containing the estrogen response element. The ERDelta4 protein was not able to activate transcription of a reporter gene. However, it inhibited estrogen-dependent transcriptional activation in a dominant negative fashion when it was cotransfected with the wild-type ER and reporter plasmid. Because it has been shown that ERDelta4 is not able to bind to its response element, the observed inhibitory effect probably occurs through protein-protein interactions. Although several variants of the ER have been described from cancerous cells, none has been identified in ovarian tissues, and ERDelta4 is the only isoform detected in normal tissues. These results may have implications for understanding the physiological role of ERDelta4 in normal cells, because it may affect the function of the wild-type ER, depending on the level of the variant ER protein relative to that of the wild-type ER.

Alternative Splicing↗

PubMed1995

The HPV16 E5 protein: expression, detection, and stable complex formation with transmembrane proteins in COS cells.

The human papillomavirus-16 (HPV16) E5 gene is able to induce stable growth transformation and transient mitogenic stimulation in a variety of cultured cell systems. To characterize the biochemical properties of the hydrophobic HPV16 E5 transforming protein, we have constructed vectors expressing the wild-type HPV16 E5 gene and have generated antipeptide antisera. The 10-kDa E5 protein was readily detectable in transfected COS monkey cells by using these antisera either for immunoprecipitation of metabolically labeled cells or for immunoblotting. Coimmunoprecipitation analysis of cells coexpressing the viral protein and various growth factor receptors demonstrated stable complex formation between the E5 protein and the epidermal growth factor receptor, platelet-derived growth factor beta receptor, colony stimulating factor-1 receptor, and p185neu. The E5 protein also formed a stable complex with the vesicular stomatitis virus glycoprotein. These experiments indicated that the HPV16 E5 protein was able to participate in complex formation with a variety of transmembrane proteins, a property which may contribute to the biological activities of the viral protein. In addition, the expression vectors and antibodies described here will be useful reagents in examining various aspects of HPV16 E5 expression and function.

Animals↗

PubMed1995

Epitope analysis of a sperm acrosomal antigen defined by HSA-5 monoclonal antibody.

Among the monoclonal antibodies recommended by the WHO Sperm Antigen Workshop for immunocontraceptive vaccine development, HSA-5 showed a high degree of sperm specificity and significantly inhibited in vitro fertilization in both humans and mice. Using a Western blot assay, HSA-5 was found to recognize a sperm antigen designated as HSAg-5 (human) or MSAg-5 (mouse) which ranged in molecular weight from 18 to 100 kDa. This monoclonal antibody was used as the probe for the immunoscreening of mouse testis cDNA libraries constructed in the lambda gt-11 expression vector. One of the positive cDNA clones was shown to have a cDNA insert of approximately 1 kb and to encode a recombinant fusion protein containing 77 amino acid residues in the C-terminal region of MSAg-5. This 1 kb cDNA insert was engineered in a pGEX vector to express a recombinant glutathione S-transferase fusion protein (GST-5). Using an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, both anti-GST-5 sera and the monoclonal antibody were shown to react with GST-5. The Northern blot of a mouse testis RNA preparation revealed that the isolated cDNA probe hybridized with a 4.0 kb mRNA. Several oligopeptides were synthesized based on the predicted C-terminal hydrophilic regions of the recombinant fusion protein. Using ELISA and a dot blot assay, peptide regions containing the immunogenic epitopes recognized by HSA-5 monoclonal antibody were identified.

Amino Acid Sequence↗

PubMed1994

Gene cloning of the human cytomegalovirus (HCMV) antigen reactive with the serum from a HCMV-infected patient.

The human cytomegalovirus(HCMV) gene encoding the protein reactive with the sera of HCMV-infected patient was cloned and characterized. A reactive phage clone was screened from a lambda gt11 expression library of cDNA of HCMV AD169 strain using HCMV-infected patient sera. The recombinant protein was expressed as 138 kDa-fusion protein with beta-galactosidase, which was reactive with IgM or IgG HCMV antibody-positive sera, but not with anti-HCMV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with HCMV AD169 sequences revealed that it was composed of 709 base pairs spanning between 0.174 and 0.177 map units of the UL32 region of the HCMV AD169 strain genome. This position corresponded to a part of the gene encoding 150 kDa phosphoprotein-(pp150), a major tegument protein, which was reported as an immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. These results suggested that pp150 was an immunogenic protein in natural HCMV infection and therefore this clone was regarded as a useful candidate for developing an antigen for the serodiagnosis of HCMV.

Antibodies, Viral↗

PubMed1993

Identification of 8-hydroxyguanine glycosylase activity in mammalian tissues using 8-hydroxyguanine specific monoclonal antibody.

Here we report the finding of glycosylase activity in mammalian tissues to remove 8-hydroxyguanine residues from DNA. To detect this activity, a synthetic duplex DNA containing an 8-hydroxyguanine residue was used as the substrate, and before the product was analyzed by HPLC-electrochemical detection (ECD), 8-hydroxyguanine released by the enzymatic action was specifically collected from reactions using immunoaffinity columns of 8-hydroxyguanine specific monoclonal antibody. With the application of immunoaffinity column chromatography, a single peak of 8-hydroxyguanine free from many interfering peaks of unknown origin was clearly demonstrated in HPLC-ECD even with crude tissue extracts used as assay materials. All the tissues of the rats examined showed the activity, suggesting its presence is ubiquitous. The present coupled technique of immunoaffinity column chromatography and HPLC-ECD will be very useful to many future studies of 8-hydroxyguanine glycosylase such as its activity detection, characterization and purification, etc.

Animals↗

PubMed1993

Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.

Bovine papillomavirus 1↗