Biomedical subjects

Zheng Tu

Publications and source records attributed to Zheng Tu.

13 recordsLinked to original sources

PubMed2006

IKKalpha regulates the mitotic phase of the cell cycle by modulating Aurora A phosphorylation.

The IKK complex includes two catalytic components, IKKalpha and IKKbeta, in addition to the scaffold protein IKKgamma/NEMO. Even though IKKalpha and IKKbeta share significant sequence homology, they have distinct biological roles with IKKbeta regulates the classical pathway of NF-kappaB activation and IKKalpha regulates the alternative pathways. In addition, it has been shown that the IKKs regulate the proliferation of both normal and tumor cells; however, the mechanisms by which the IKKs regulate the cell cycle remain to be further defined. Here, we demonstrate that IKKalpha, but not IKKbeta, has role in regulating the M phase of the cell cycle. IKKalpha siRNA knock -down resulted in increased numbers of cells in the G(2)/M phase of the cell cycle as compared to control and IKKbeta siRNA transfected HeLa cells. This effect was associated with upregulation of cyclin B1 and Plk1 protein levels and increased histone H3 phosphorylation, consistent with a potential role of IKKalpha in the regulation of M phase regulatory factors. IKKalpha was found to be associated with Aurora A in the centrosome and regulate Aurora A phosphorylation at threonine residue 288, a site which is important in modulating its kinase activity. Taken together, these data provide the evidence that IKKalpha regulates the M phase of the cell cycle by modulating Aurora A phosphorylation and activation leading to the regulation of the M phase of the cell cycle.

Aurora Kinases↗

PubMed2006

17 beta-estradiol activates PI3K/Akt signaling pathway by estrogen receptor (ER)-dependent and ER-independent mechanisms in endometrial cancer cells.

Cellular response to estrogen is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transducing pathways. The main aims are to study if PI3K/Akt signaling pathway can be activated by 17beta-estradiol (E2) via non-nuclear action and to investigate the relationship of the action of E2 and ER in endometrial cancer cells expressing with different status of ER. The levels of phosphorylated Akt (Ser473) (P-Akt) and total Akt were examined by western blot and Akt kinase activity was measured in cells after stimulation with 1 microM E2 at different time points. Inhibitory role of LY294002 on activation of Akt induced by E2 and its estrogen antagonist, ICI182780 were also tested. P-Akt/Akt was used as a measure of activation of Akt. We found that maximum P-Akt/Akt and Akt kinase activity took place at 30 min in Ishikawa cells and 15 min in HEC-1A cells and the activation persisted for at least 2 h after stimulation with 1 microM E2. The activation of Akt elicited gradually with increasing doses of E2. PI3K inhibitor, LY294002, stopped the activating Akt in a dose-dependent manner and 50 microM LY294002 completely blocked the activation of Akt induced by E2. ICI182780 could block the activation of PI3K/Akt in ER-positive Ishikawa cells but not in HEC-1A cells with poor-expressed ER. This study demonstrated that E2 is able to promptly activate PI3K/Akt signal pathway in Ishikawa cells in an ER-dependent manner and ER-independent in HEC-1A cells. Blockage of PI3K/Akt cascade may become a potential and effective way to control endometrial carcinoma, especially in ER-negative cancers, which show no response to endocrinal therapy.

Cell Line, Tumor↗

PubMed2006

IKK alpha regulates estrogen-induced cell cycle progression by modulating E2F1 expression.

The IkappaB kinase (IKK) complex consists of the catalytic subunits IKKalpha and IKKbeta and a regulatory subunit, IKKgamma/NEMO. Even though IKKalpha and IKKbeta share significant sequence similarity, they have distinct biological roles. It has been demonstrated that IKKs are involved in regulating the proliferation of both normal and tumor cells, although the mechanisms by which they function in this process remain to be better defined. In this study, we demonstrate that IKKalpha, but not IKKbeta, is important for estrogen-induced cell cycle progression by regulating the transcription of the E2F1 gene as well as other E2F1-responsive genes, including thymidine kinase 1, proliferating cell nuclear antigen, cyclin E, and cdc25A. The role of IKKalpha in regulating E2F1 was not the result of reduced levels of cyclin D1, as overexpression of this gene could not overcome the effects of IKKalpha knock-down. Furthermore, estrogen treatment increased the association of endogenous IKKalpha and E2F1, and this interaction occurred on promoters bound by E2F1. IKKalpha also potentiated the ability of p300/CBP-associated factor to acetylate E2F1. Taken together, these data suggest a novel mechanism by which IKKalpha can influence estrogen-mediated cell cycle progression through its regulation of E2F1.

Animals↗

PubMed2006

[Genetic polymorphism of penta e locus in four Chinese nationalities].

To study the genetic polymorphism of Penta E locus in four Chinese nationalities using home made reagent kits, DNA samples were obtained from about 400 unrelated peoples of four different Chinese nationalities. As a result, we found 20 alleles in the four nationalities with frequencies ranging from 0.0048 to 0.2396. The genotype frequencies of Penta E locus met Hardy-Weinberg equilibrium. It proved that Penta E locus was a high polymorphic STR genetic marker and was valuable for forensic science.

Asian People↗

PubMed2005

Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

OBJECTIVE: To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. METHODS: We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. RESULTS: [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P < 0.05). More colonies were grown 10 days after incubating pcDI-[12Asp]-K-ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. CONCLUSIONS: K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

Animals↗

PubMed2004

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

OBJECTIVE: To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. METHODS: (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. RESULTS: (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P < 0.01) and 1.9-fold (128 +/- 37, 349 +/- 30, P < 0.05) in ERalpha and ERbeta, respectively, in pcDI-[(12)Asp]K-ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P < 0.05) and 1.8:1 (493 +/- 20, 284 +/- 20, P < 0.01), respectively; In HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P < 0.01) and 2.4:1 (405 +/- 33, 165 +/- 15, P < 0.01), respectively. (3) In low serum (2%) culture condition, estradiol (E(2)) stimulated luciferase activity with an increase of 13-fold (130 +/- 42, 1681 +/- 242, P < 0.01) in pcDI-[(12)Asp] K-ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P < 0.001) in HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P < 0.01) and 6-fold (2148 +/- 259, 12,705 +/- 2670, P < 0.001), respectively. rafS621A mutant had suppressive effects on luciferase activities in HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P < 0.05) and 6.4:1 (1949 +/- 212, 304 +/- 60, P < 0.01), respectively. CONCLUSIONS: (1) [(12)Asp]K-ras4B can enhance the expression of ERalpha and beta proteins. This may be correlated with [(12)Asp]K-ras4B/raf signaling pathway. (2) The effect of mutant-type [(12)Asp]K-ras4B gene on ERs transcriptional activity in HEC-1A cells appears to need E(2).

Animals↗

PubMed2004

A simple and efficient method for extracting DNA from old and burned bone.

It has been a challenge to extract DNA from bones previously soaked in water, burned, or buried for a long time, due to the reduced quality and quantity of DNA in the bone samples. The dramatic degradation of the DNA and the presence of PCR inhibitors in the collagen significantly complicate the process of DNA identification in dated and charred bones. In this article, we present a novel strategy to obtain DNA from bones based on the use of cetyltrimethylammonium bromide (CTAB) lysis buffer and isoamyl alcohol-chloroform extraction with subsequent DNA purification using the DNA IQ System, or alternatively the QIAquick system. When applied to bones soaked, burned or buried for up to nine years, this method increases the purity and yield of DNA with respect to the traditional phenol-chloroform method and significantly improves multiplex STR genotyping using fluorescence-based methods. The results of this research will assist forensic scientists in the identification of DNA from victims whose bodies underwent significant trauma or burning, precluding the utilization of traditional forensic DNA identification techniques.

Bone and Bones↗

PubMed2003

Inhibiting the expression of DNA replication-initiation proteins induces apoptosis in human cancer cells.

DNA replication-initiation proteins are expressed in cancer cells, whereas some of these proteins are not expressed in nonproliferating normal cells. Therefore, replication-initiation proteins may present attractive targets for anticancer therapy. Using selected antisense oligodeoxynucleotides and small interfering RNA molecules targeted to the mRNA encoding the DNA replication-initiation proteins hCdc6p, hMcm2p, and hCdc45p, we show that the target genes could be effectively and specifically silenced and that, consequently, DNA replication and cell proliferation were inhibited in cultured human cells. In addition, silencing of these genes resulted in apoptosis in both p53-positive and -negative cancer cells but not in normal cells: cancer cells entered an abortive S-phase, whereas normal cells arrested mainly in G(1) phase. Our studies are the first to suggest that inhibiting the expression of selective replication-initiation proteins is a novel and effective anticancer strategy.

Animals↗

PubMed2003

Subdomain VIII is a specificity-determining region in MEKK1.

MAPK/ERK kinase kinase 1 (MEKK1) is a mitogenactivated protein kinase kinase kinase (MAP3K) of the stress-induced JNK pathway. Once activated, MEKK1 phosphorylates the MAP2K MKK4, which in turn phosphorylates JNK. MEKK1 also has the capacity to activate IKK, the central protein kinase of the NF-kappa B pathway. The molecular determinants responsible for the ability of MEKK1 to recognize specific substrates are poorly understood. We report here that select point mutations in subdomain VIII of the protein kinase domain of MEKK1 (MEKK1 Delta) differentially affect its ability to activate MKK4 and IKK, and consequently AP1 and NF-kappa B reporter genes. Moreover, binding of MKK4 to MEKK1 Delta protects the latter from cleavage at an engineered protease target site in subdomain VIII. Collectively these results provide evidence that subdomain VIII of MEKK1 is involved not only in binding to, but also in discrimination of, protein substrates.

Amino Acid Sequence↗

PubMed2003

Mutations in protein kinase subdomain X differentially affect MEKK2 and MEKK1 activity.

MAPK/ERK kinase kinase 2 (MEKK2) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of protein kinases. MAP3Ks are components of a three-tiered protein kinase pathway in which a MAP3K phosphorylates and activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates a mitogen-activated protein kinase (MAPK). We have previously identified residues within protein kinase subdomain X in the MAP3K, MEKK1, that are critical for its interaction with the MAP2K, MKK4, and MEKK1-induced MKK4 activation. We report here that kinase subdomain X also plays a critical role in MEKK2 activity. Select point mutations in subdomain X impair MEKK2 phosphorylation of the MAP2Ks, MKK7 and MEK5, abolish MEKK2-induced activation of the MAPKs, JNK1 and ERK5, and diminish MEKK2-dependent activation of an AP-1 reporter gene. Interestingly, the spectrum of mutations in subdomain X of MEKK2 that affects its activity is overlapping with but not identical to those that have effects on MEKK1. Thus, mutations in subdomain X differentially affect MEKK2 and MEKK1.

Amino Acid Sequence↗

PubMed2003

A subdomain of MEKK1 that is critical for binding to MKK4.

Mitogen-activated protein kinase (MAPK) cascades are central components of signal transduction pathways induced by mitogens and stresses. They consist of a three-kinase module in which a mitogen-activated protein kinase kinase kinase (MAP3K) activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates MAPK. The molecular determinants that underlie specific MAP3K-MAP2K interactions are poorly understood. In this study, we examined the interaction between the MAP3K MEKK1 and MKK4, a MAP2K of the JNK pathway. Select point mutations in subdomain X of the catalytic domain of MEKK1 (MEKK1delta) were found to impair the ability of MEKK1delta to bind to and activate MKK4. Such mutations were also found to impair MEKK1delta-induced activation of an AP1 reporter gene. These studies point to a critical role for subdomain X in the interaction of MEKK1 with MKK4.

Amino Acid Sequence↗

PubMed2002

[Regulation of human progesterone receptor isoforms A and B in uterine endometrial carcinoma by estrogen and insulin-like growth factor-1].

OBJECTIVE: To investigate the regulation of human progesterone receptor isoforms A and B in uterine endometrial carcinoma by estrogen and insulin-like growth factor-1 (IGF-I) so as to provide theoretical basis for clinical hormone treatment of uterine endothelium cancer. METHODS: The uterine endometrial adenocarcinoma cell line HEC-IB and the breast cancer cell line MCF-7 were cultured in vitro. The HEC-IB cells were stimulated by 10 nmol/L estrogen and 20 ng/ml IGF-I respectively for 72 h. The MCF-7 cells were stimulated by 10 nmol/L estrogen for 72 h. Western blotting was used to examine the protein levels of the two isoforms of receptors of progesterone. RNA was extracted from the cells. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of the isoform B. RESULTS: (1) Western blotting showed that the photodensity values of hPRA and hPRB did not change significantly after the HEC-IB cells were stimulated by estrogen for 72 hours (P = 0.716, and 0.391 respectively), however, the hPRA and hPRB were significantly down-regulated after IGF-I had been given for 72h (P = 0.008 and 0.002 respectively). After MCF-7 cell was given estrogen for 72 h hPR-A tended to be up- regulated but this change had no significant difference (P = 0.074) however, hPRB was significantly up- regulated (P = 0.044). (2) RT-PCR showed that hPRB mRNA was not expressed in HEC-IB cells originally, and became positive after stimulation by estrogen and IGF-1 for 72 hours respectively, however, the expression level being higher in the HEC-IB cells stimulated by estrogen than in those stimulated by IGF-1. hPRB was not expressed in MCF-7 cells originally too, and became positive after simulation by estrogen and IGF-1 for 72 hours respectively, however, the expression level being higher in the MCF-7 cells stimulated by estrogen than in those stimulated by IGF-1. CONCLUSION: (1) Estrogen and IGF-I regulate hPR isoforms and have cell specialty. (2) Estrogen and IGF-I up- regulate hPRB mRNA at gene level or transcription level, but there are some inhibitory factors which make the protein production become less after transcription.

Dinoprostone↗

PubMed2002

[Time-dependent and dose-dependent regulation of human progesterone receptor isoforms A and B in uterine endometrial carcinoma by human insulin-like growth factor-I].

OBJECTIVE: We study the regulation of human progesterone receptor isoforms A and B in uterine endometrial carcinoma cell line by different concentration of human insulin-like growth factor-I (IGF-I) for different time, to investigate the roles of IGF-I and progesterone receptor isoforms in uterine endometrial carcinoma. METHODS: The uterine endometrial adenocarcinoma cell line HEC-IB was cultured in vitro and the breast cancer cell line MCF-7 was used as control. Western blot was applied to examine the changes of the two isoforms by different concentration IGF-I for different time. RESULTS: (1) In HEC-IB cell line, 10 ng/ml IGF-I made hPRB up-regulated in the first 24 h. But according to lager concentration and longer time, human progesterone receptor (hPR) B became down-regulated, which were significant at 20 ng/ml IGF-I for 72 h and 40 ng/ml IGF-I for 48 - 72 h. The change of hPRA was like hPRB. (2) In MCF-7 cell line, 10 ng/ml and 40 ng/ml IGF-I made hPRA and hPRB significantly up-regulated in 24, 48, 72 h. Twenty ng/ml IGF-I made hPRB up-regulated also in the first 24 h. But in 48 h and 72 h, down-regulation of hPRB was detected. Twenty ng/ml IGF-I made hPRA down-regulated in 24, 48, 72 h. CONCLUSIONS: (1) The regulation of IGF-I to hPR isoforms has cell-type specific and dose-dependent and time-dependent. (2) In HEC-IB cell line, 10 ng/ml IGF-I made hPRB significantly up-regulated in 24 h. But following exposure to IGF-I at larger concentration and longer time, hPRB became down-regulated. The change of hPRA is like hPRB.

Blotting, Western↗