Biomedical subjects

Hoyun Lee

Publications and source records attributed to Hoyun Lee.

14 recordsLinked to original sources

PubMed2006

[Chloroquine increased radiosensitivity of radioresistant MDA-MB 231 cells and its molecular mechanism].

OBJECTIVE: To study the genes associated with CQ (Chloroquine) increased radiosensitivity in radioresistant MDA-MB 231 cells. METHODS: The survival curve of MDA-MB 231 breast cancer cells was establish in the presence or absence of CQ after irradiation by colongenic assay. The relationship between certain protein expressions (MLH1, ERCC1, PARP, RAD51, XRCC4) and CQ increased radiosensitivity in radioresistant MDA-MB 231 cells was examined by Western blot method. The genes which are associated with CQ increased radiosensitivity were studied by microarray analysis. RESULTS: CQ increased deaths of parental MDA-MB 231 cells, radiosensitive MDA-MB 231 cells and radioresistant MDA-MB 231 cells were compared with those of the CQ-untreated control cells after irradiation, and the Radiation Potentiation Factor were 1.21-1.23. The expression of ERCC1 in the presence and absence of CQ in the radioresistant cells was significantly higher than that of sham-irradiated control cells by Western blot method. After irradiation, RAD51 level was increased up to 3-hour radiation and then gradually reduced. The expression of MLH1 was lower in 24 hours than that of other time points after irradiation. There were no differences on the expressions of MLH1, ERCC1, RAD51 in cells with and without CQ, which suggested that these genes weren't correlated with CQ increased radiosensitivity. XRCC4 and PARP levels were not changed by irradiation. Microarray analysis showed that TNP3, FRIH, CA21, ADTA genes might be related to CQ increased radiosensitivity in radioresistant cells. CONCLUSION: CQ can increase radiosensitivity in radioresistant MDA-MB 231 breast cancer cells. It is not related to MLH1, ERCC1, PARP, RAD51, XRCC4 genes. TNP3 may play an important role in CQ increase radiosensitivity in radioresistant MDA-MB 231 breast cancer cells.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Importin-beta mediates Cdc7 nuclear import by binding to the kinase insert II domain, which can be antagonized by importin-alpha.

We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-beta binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-alpha and -beta bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-beta is approximately 10 times higher than for importin-alpha at low protein concentrations of an equimolar ratio. Immunodepletion of importin-beta, but not importin-alpha, abrogates Cdc7 nuclear import, and the addition of importin-beta to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-beta, but not anti-importin-alpha antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-alpha to Cdc7. Further studies by site-directed mutagenesis suggest that Lys306 and Lys309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization.

Active Transport, Cell Nucleus↗

PubMed2006

Characterization of the human proliferating cell nuclear antigen physico-chemical properties: aspects of double trimer stability.

Its toroidal structure allows the proliferating cell nuclear antigen (PCNA) to wrap around and move along the DNA fiber, thereby dramatically increasing the processivity of DNA polymerization. PCNA is also involved in the regulation of a wide spectrum of other biological functions, including epigenetic inheritance. We have recently reported that mammalian PCNA forms a double trimer complex, which may be critically important in coordinating DNA replication and other cellular functions. To gain a better understanding of the stability of PCNA complexes, we characterized the physico-chemical properties of the PCNA structure by in vivo and in vitro approaches. The data obtained by gel filtration and nondenaturing gel electrophoresis of native PCNA molecules confirm our previous observations, obtained using formaldehyde crosslinking, in which PCNA exists in the cell as a double trimer. We have also found that optimal pH (pH 6.5-7.5) is critical for the stability of the PCNA structure. The presence or absence of ATP, dithiothreitol, and Mg2+ does not affect the stability of the PCNA trimer or double trimer. However, 0.02% SDS can effectively inhibit PCNA double trimer, but not single trimer, formation. Interestingly, glycerol and ammonium sulfate significantly destabilize both PCNA trimer and double trimer structures.

Chromatography, Gel↗

PubMed2005

High levels of Cdc7 and Dbf4 proteins can arrest cell-cycle progression.

Cdc7-Dbf4 serine/threonine kinase is essential for initiation of DNA replication. It was previously found that overexpression of certain replication proteins such as Cdc6 and Cdt1 in fission yeast resulted in multiple rounds of DNA replication in the absence of mitosis. Since this phenomenon is dependent upon the presence of wild-type Cdc7/Hsk1, we hypothesized that high levels of Cdc7 and/or Dbf4 could also cause multiple rounds of DNA replication, or could facilitate entry into S phase. To test this hypothesis, we transiently overexpressed hamster Cdc7, Dbf4 or both in CHO cells. Direct observations of individual cells by fluorescence microscopy and flow cytometric analysis on cell populations suggest that overexpression of Cdc7 and/or Dbf4 does not result in multiple rounds of DNA replication or facilitating entry into S phase. In contrast, moderately increased levels of Dbf4, but not Cdc7, cause cell-cycle arrest in G2/M. This G2/M arrest coincides with hyperphosphorylation of Cdc2/Cdk1 at Tyr-15, raising the possibility that high levels of Dbf4 may activate a G2/M cell-cycle checkpoint. Further increase in Cdc7 and/or Dbf4 by 2-4 fold can arrest cells in G1 and significantly slow down S-phase progression for the cells already in S phase.

Animals↗

PubMed2005

Complete condensation in a zero range process on scale-free networks.

We study a zero range process on scale-free networks in order to investigate how network structure influences particle dynamics. The zero range process is defined with the rate p(n) = n(delta) at which particles hop out of nodes with n particles. We show analytically that a complete condensation occurs when delta < or = delta(c) triple bond 1/(gamma-1) where gamma is the degree distribution exponent of the underlying networks. In the complete condensation, those nodes whose degree is higher than a threshold are occupied by macroscopic numbers of particles, while the other nodes are occupied by negligible numbers of particles. We also show numerically that the relaxation time follows a power-law scaling tau approximately L(z) with the network size L and a dynamic exponent z in the condensed phase.

Journal Article↗

PubMed2005

Proliferating cell nuclear antigen (PCNA) may function as a double homotrimer complex in the mammalian cell.

The diverse function of proliferating cell nuclear antigen (PCNA) may be regulated by interactions with different protein partners. Interestingly, the binding sites for all known PCNA-associating proteins are on the outer surface or the C termini ("front") sides of the PCNA trimer. Using cell extracts and purified human PCNA protein, we show here that two PCNA homotrimers form a back-to-back doublet. Mutation analysis suggests that the Arg-5 and Lys-110 residues on the PCNA back side are the contact points of the two homotrimers in the doublet. Furthermore, short synthetic peptides encompassing either Arg-5 or Lys-110 inhibit double trimer formation. We also found that a PCNA double trimer, but not a homotrimer alone, can simultaneously accommodate chromatin assembly factor-1 and polymerase delta. Together, our data supports a model that chromatin remodeling by chromatin assembly factor-1 (and, possibly, many other cellular activities) are tightly coupled with DNA replication (and repair) through a PCNA double trimer complex.

Animals↗

PubMed2005

NFATc3 deficiency may contribute to the development of mammary gland adenocarcinoma in aging female mice.

The nuclear transcription factor of activated T cells (NFAT) suggested to be a tumor suppressor. Here we report that two out of three NFATc3-/- and two in four NFATc3 +/- female mice developed aggressive mammary adenocarcinoma by 12.5 and 16 mo of age, respectively, with no occurrences in age-matched wild-type littermates (N-14). Thus, our data suggest that NFATc3 can suppress the development of mammary gland tumors in female mice.

Adenocarcinoma↗

PubMed2005

Chloroquine-mediated radiosensitization is due to the destabilization of the lysosomal membrane and subsequent induction of cell death by necrosis.

The anti-malarial drug chloroquine (CQ) is also thought to be a potential radiation sensitizer. To gain a better understanding of how the lysomotropic CQ can potentiate the effects of ionizing radiation, we investigated the effects of CQ on lysosomal and mitochondrial membrane stability, the subcellular localization of ceramide, plasma membrane permeability, and the mode of cell death in response to irradiation. We found that CQ accumulated in the lysosomes and thus lysosomal volumes increased. As a result, both the lysosomal and plasma membranes were destabilized. After 7 Gy irradiation, most ceramide was associated with the lysosomes in the cells treated with CQ but not in the CQ-untreated control. The elevated levels of ceramide in the lysosomes of the CQ-treated cells appeared to further destabilize the lysosomal and plasma membranes of the cell. Both CQ-treated and -untreated cells had approximately the same rate of cell death by apoptosis after 7 Gy irradiation (P > 0.05, ns). However, in contrast to the CQ-untreated control, the CQ-treated cells underwent massive cell death by necrosis at 24-48 h after irradiation (P < 0.05). Taken together, our data support the idea that the increase in cytotoxic effects by the combination of CQ and radiation is due to radiation-mediated apoptosis and CQ-mediated necrosis.

Apoptosis↗

PubMed2004

Analytic study of the three-urn model for separation of sand.

We present an analytic study of the three-urn model for separation of sand, which can be regarded as a zero-range process. We solve analytically the master equation and the first-passage problem. We find that the stationary probability distribution obeys the detailed balance and is governed by the free energy. We find that the characteristic lifetime of a cluster diverges algebraically with exponent 1/3 at the limit of stability. We also give a general argument that the scaling behavior is robust with respect to different expressions of the flux.

Journal Article↗

PubMed2004

The post-translational modifications of proliferating cell nuclear antigen: acetylation, not phosphorylation, plays an important role in the regulation of its function.

The diverse function of proliferating cell nuclear antigen (PCNA) is thought to be due, in large part, to post-translational modifications. Here we show by high resolution two-dimensional PAGE analysis that there are three distinct PCNA isoforms that differ in their acetylation status. The moderately acetylated main (M) form was found in all of the subcellular compartments of cycling cells, whereas the highly acetylated acidic form was primarily found in the nucleoplasm, nuclear matrix, and chromatin. Interestingly, the deacetylated basic form was most pronounced in the nucleoplasm of cycling cells. The cells in G(0) and the cytoplasm of cycling cells contained primarily the M form only. Because p300 and histone deacetylase (HDAC1) were co-immunoprecipitated with PCNA, they are likely responsible for the acetylation and deacetylation of PCNA, respectively. We also found that deacetylation reduced the ability of PCNA to bind to DNA polymerases beta and delta. Taken together, our data support a model where the acidic and M forms participate in DNA replication, whereas the basic form is associated with the termination of DNA replication.

Acetylation↗

PubMed2003

Controlling chaos to solutions with complex eigenvalues.

We derive formulas for parameter and variable perturbations to control chaos using linearized dynamics. They are available irrespective of the dimension of the system, the number of perturbed parameters or variables, and the kinds of eigenvalues of the linearized dynamics. We illustrate this using the two coupled Duffing oscillators and the two coupled standard maps.

Journal Article↗

PubMed2003

Analytic study of the urn model for separation of sand.

We present an analytic study of the urn model for separation of sand recently introduced by Lipowski and Droz [Phys. Rev. E 65, 031307 (2002)]. We solve analytically the master equation and the first-passage problem. The analytic results confirm the numerical results obtained by Lipowski and Droz. We find that the stationary probability distribution and the shortest one among the characteristic times are governed by the same free energy. We also analytically derive the form of the critical probability distribution on the critical line, which supports their results obtained by numerically calculating Binder cumulants (A. Lipowski and M. Droz, e-print cond-mat/0201472).

Journal Article↗

PubMed2003

Observation of multiple isoforms and specific proteolysis patterns of proliferating cell nuclear antigen in the context of cell cycle compartments and sample preparations.

The proliferating cell nuclear antigen (PCNA) is an essential component for eukaryotic chromosomal DNA replication and repair. PCNA forms a homotrimer ring, which may function as a DNA sliding clamp for DNA polymerases and, possibly, a docking station for other replication- and repair-related proteins. Several reports have suggested the existence of different PCNA isoforms. Here we confirm, using high resolution two-dimensional electrophoresis with narrow pH ranges, the existence of three PCNA isoforms in both Chinese hamster and human breast cancer cells. Among the three isoforms, M or main form is the dominant one throughout the cell cycle while the relative amounts of the minor components A (acidic) and B (basic) forms appear to vary during the cell cycle. We also observed that a specific pattern of PCNA proteolysis occurred during isoelectric focusing in spite of high urea (8 M) and detergent (2% 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate), which was largely inhibited by the proteosome inhibitor MG132 or boiling. Interestingly, the proteolysis pattern was mainly observed with samples isolated from cells in S and G2 phases. A similar but much lower level of PCNA proteolysis also occurred in vivo within the nuclei of the cells in S phase. Taken together, our data are consistent with the idea that the existence of the different isoforms and specific proteolysis of PCNA are relevant to its functions in vivo.

Animals↗

PubMed2002

Human Dbf4/ASK promoter is activated through the Sp1 and MluI cell-cycle box (MCB) transcription elements.

Dbf4 is the regulatory subunit of Cdc7 kinase, which is essential for entry into and traversing through S phase. The level of Dbf4, which is critical for the activation of Cdc7, is regulated by transcription and protein degradation. To gain a better understanding as to how the transcription of human Dbf4 (HuDbf4) is regulated, we have cloned and characterized its promoter. We found that HuDbf4 core promoter is localized within (-)211 to -285 of the translation start-codon. This 75 bp DNA segment contains, among others, a putative MluI Cell-cycle Box (MCB). A point mutation within the MCB dramatically reduced the promoter activity. This is the first example that an MCB element plays an essential role in the activation of a core promoter in mammalian cells. The auxiliary elements required for the full promoter activity are present within 162-bp upstream from the core promoter (i.e., -286/-447). A point mutation within the Sp1 element at -353/-361 resulted in a decrease of promoter activity to the basal level, while the deletion of the putative HES-1 at -326/-331 dramatically increased the promoter activity. Taken together, our data suggests that the MCB element is essential for the core promoter activation, while the Sp1 positive regulator and the HES-1 repressor coordinately determine the efficiency of the HuDbf4 promoter. We have also found: (i) that the major transcription initiations occur at -220, -235 and -245; (ii) that HuDbf4 gene consists of 12 exons, which spread over a 33-kb region.

Bacterial Proteins↗