Biomedical subjects

R P Huang

Publications and source records attributed to R P Huang.

At least 19 recordsLinked to original sources

PubMed2001

Detection of multiple proteins in an antibody-based protein microarray system.

A new antibody-based protein array assay is described. This assay combines the advantages of the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL) and high-throughput of microspot. In this system, the capture proteins, either antibodies or antigens are spotted onto membranes in an array format. Biological samples are then incubated with membranes. After antigens or antibodies in the samples bind to their corresponding targets and unbound proteins are washed away, the membranes are exposed to Horseradish Peroxidase (HRP)-conjugated antibody(ies). The signals are finally visualized with ECL system. Experiments demonstrate that multiple cytokines and antibodies can be simultaneously detected using this new approach. The procedure is so simple that no sophisticated equipment is required. The concept should be able to be extended to develop a high-throughput protein array system. Future applications of this new approach include direct protein expression profiling, immunological disease diagnostics and discovery of new biomarkers.

Antibodies↗

PubMed2001

Simultaneous detection of multiple cytokines from conditioned media and patient's sera by an antibody-based protein array system.

We have developed a novel technique for high-throughput simultaneous screening of multiple cytokine expression based on a protein array system. Our method has the advantage of showing the specificity of enzyme-linked immunosorbent assays, sensitivity of enhanced chemiluminescence (ECL), and high-throughput of microspot. In this system, the cytokine array membranes were created by spotting capture antibodies onto the membranes. The membranes were then incubated with biological samples such as conditioned media and patient's sera. The bound proteins were then recognized by biotin-conjugated antibodies and detected by horseradish peroxidase-conjugated streptavidin coupled with ECL. Experiments demonstrated that 24 cytokines from conditioned media and patient's sera could be simultaneously detected using this new approach. This methodology should allow us to develop many high-density protein array systems to detect a variety of proteins. To validate and quantitate the expression of key molecules in a wide range of samples, we have developed conditioned medium arrays to evaluate hundreds and even thousands of samples from individual cells and patients in a single microarray. The combinations of protein arrays and conditioned medium arrays or serum arrays will provide a powerful tool to identify the protein expression profiles and rapidly validate their expression in many types and numbers of samples.

Antibodies↗

PubMed2001

Connexin 43 (cx43) enhances chemotherapy-induced apoptosis in human glioblastoma cells.

Stable re-expression of connexin 43 (cx43) in human glioblastoma suppresses transformation and tumorigenicity. The present study was designed to examine the role of cx43 in chemotherapy-induced apoptosis. Expression of cx43 in human glioblastoma cells significantly increased sensitivity to several common chemotherapeutic agents, including etoposide, paclitaxel (Taxol) and doxorubicin, compared with control-transfected cells. The increased sensitivity to chemotherapeutic agents resulted from apoptosis as evidenced by Hoechst dye staining, TUNEL assay and annexin V assay. These cx43-mediated effects were coupled with decreased expression of the specific apoptosis inhibitor bcl-2. Over-expression of bcl-2 in cx43-transfected cells partially confers the resistance to apoptosis induced by etoposide, suggesting that the cx43-mediated apoptosis to chemotherapeutic agents is regulated in part through the down-regulation of bcl-2 expression. Furthermore, the cx43-mediated apoptosis in response to chemotherapeutic drugs may not be linked to increased gap junctional communication in cx43-transfected cells. Our results demonstrate a new role of cx43 in the mediation of apoptosis during chemotherapy.

Antineoplastic Agents↗

PubMed2001

Hydrogen peroxide promotes transformation of rat liver non-neoplastic epithelial cells through activation of epidermal growth factor receptor.

Previous studies demonstrated that hydrogen peroxide (H(2)O(2)) is a tumor promoter in the rat liver epithelial cell line T51B. We investigated the pathway linking H(2)O(2) to tumor promotion. H(2)O(2) can directly induce tyrosine phosphorylation of epidermal growth factor receptor (EGFR). H(2)O(2) and epidermal growth factor exerted similar effects on the induction of early growth response genes, disruption of gap junction communication, triggering of calcium inflow, and promotion of transformation. Furthermore, the effect of H(2)O(2) on tumor promotion was blocked by abrogation of EGFR activation. Our results suggested that tumor promotion by H(2)O(2) is mediated mainly through activation of EGFR in T51B cells.

Animals↗

PubMed2001

Enhanced apoptosis under low serum conditions in human glioblastoma cells by connexin 43 (Cx43).

Connexin 43 (Cx43), a structural component of gap junctions, is believed to function as a tumor suppressor gene. Previously, we showed that expression of Cx43 suppresses cell proliferation and tumorigenicity of human glioblastoma cells [Huang et al., Cancer Res 58:5089-5096, 1998] and enhances apoptosis in response to chemotherapeutic agents [Huang et al., Int J Cancer 92:130-138, 2001]. In the present study, we demonstrated that expression of Cx43 in human glioblastoma cells potentiated an apoptotic program under low-serum conditions. The Cx43-mediated effect was coupled with a decreased expression of the specific apoptosis-inhibitor bcl-2. Overexpression of bcl-2 in Cx43-transfected cells conferred resistance to apoptosis induced under low-serum conditions, suggesting that the Cx43-mediated apoptosis under low-serum conditions is regulated, in part, through the downregulation of bcl-2 expression. Furthermore, application of the phosphatidylinositol-3'-OH kinase inhibitor LY294002 specifically induced apoptosis in Cx43-transfected cells. Our results demonstrate a new role of Cx43 in the mediation of apoptosis under low serum conditions.

Apoptosis↗

PubMed2001

Simultaneous detection of multiple proteins with an array-based enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescence (ECL).

Protein arrays hold a promise in basic and clinical applications. As the first step to develop such array system, I used an array-based enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescence (ECL) to demonstrate the feasibility of simultaneous detection of multiple proteins. In the direct ELISA system, different known immunoglobulin Gs (IgGs) were immobilized onto polyvinylidine difloride (PDVF) membrane through 96-well format Bio-Dot unit. The antigens were then individually and collectively detected by incubation of membranes with different antibodies coupled with ECL. In the sandwich ELISA system, the cytokine capture antibodies were immobilized onto PDVF membranes. The membranes were then incubated with single cytokine or a combination of different cytokines. The captured cytokines were detected by biotin-conjugated antibodies coupled with ECL system. Experiments demonstrated that multiple IgGs and cytokines could be simultaneously detected using this approach with high specificity and sensitivity. More importantly, cytokines from biological samples were detected using this approach, which can be used in any general laboratory setting without any sophisticated equipment. This concept could be extended to develop a protein-based array system.

Animals↗

PubMed1999

p53 and Egr-1 additively suppress transformed growth in HT1080 cells but Egr-1 counteracts p53-dependent apoptosis.

The human fibrosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFbeta1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The effect of coexpression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by different pathways, although each additively increased the level of p21WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting effects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFbeta1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival effect of Egr-1 was dominant over the apoptotic effect of p53.

Apoptosis↗

PubMed1999

UV irradiation upregulates Egr-1 expression at transcription level.

UVC irradiation rapidly and strongly induces protein expression of the early growth response-1 gene (Egr-1) encoding a transcription factor which may have a protective function against UV damage. In this paper, we further investigate mechanisms responsible for such induction. We show that UVC irradiation also induced Egr-1 mRNA expression, increased transcription rate by nuclear run-on assay and stimulated Egr-1 promoter activity by CAT assay. The Egr-1 mRNA stability remained unchanged in UVC-treated cells. On the other hand, UVC irradiation slightly extended Egr-1 protein half-life. The induction of Egr-1 by UVC was observed in many different cell types. UVA and UVB also strongly induced Egr-1 expression. These results indicate that UVC regulates Egr-1 expression at transcription level. The induction pattern of Egr-1 by UV suggests the importance of Egr-1 in the UV response.

3T3 Cells↗

PubMed1999

The transcription factor EGR-1 suppresses transformation of human fibrosarcoma HT1080 cells by coordinated induction of transforming growth factor-beta1, fibronectin, and plasminogen activator inhibitor-1.

Re-expression of EGR-1 in fibrosarcoma HT1080 suppresses transformation including tumorigenicity (Huang, R.-P., Liu, C., Fan, Y., Mercola, D., and Adamson, E. (1995) Cancer Res. 55, 5054-5062) owing in part to up-regulation of the transforming growth factor (TGF)-beta1 promoter by EGR-1 which suppresses growth by an autocrine mechanism (Liu, C., Adamson, E., and Mercola, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11831-11836). Here we show that enhanced cell attachment contributes to the suppression via increased secretion of fibronectin (FN) and also of plasminogen activator inhibitor-1 (PAI-1). The secretion of FN and PAI-1 is strongly correlated with EGR-1 expression (RPEARSON = 0.971 and 0. 985, respectively). Addition of authentic TGF-beta1 to parental cells greatly stimulated secretion of PAI-1 but not FN, whereas addition of TGF-beta antibody or lipofection with specific antisense TGF-beta1 oligonucleotides to EGR-1-regulated cells completely inhibits the secretion of PAI-1 but not FN. However, in gel mobility shift assays pure EGR-1 or nuclear extracts of EGR-1-regulated cells specifically bind to two GC-rich elements of the human FN promoter at positions -75/-52 and -4/+18, indicating that the increased secretion of FN is likely due to direct up-regulation by EGR-1. Moreover, adhesion was greatly enhanced in EGR-1-regulated cells and was reversed by treatment with Arg-Gly-Asp (RGD) or PAI-1 antibody indicating that the secreted proteins are functional. We conclude that EGR-1 regulates the coordinated expression of gene products important for cell attachment ("oikis" factor) and normal growth control.

Cell Adhesion↗

PubMed1999

Impaired expression and posttranslational processing of connexin43 and downregulation of gap junctional communication in neoplastic human prostate cells.

BACKGROUND: Gap junctional communication (GJC) has been implicated in the control of cell proliferation. Numerous cancer cells show a decrease or loss of GJC compared to their normal counterparts. Lack of adequate information on the status of gap junctions during prostate neoplasia prompted us to examine this form of cancer, which comprises about 14% of male cancer deaths in America. METHODS: Cultured normal human prostate epithelial cells and several different human prostate tumor lines were used in this study. GJC was assayed by dye transfer, whereas Western blot and immunofluorescence methods were used to examine connexin43 (Cx43) levels and the presence of gap junctions, respectively. RESULTS: Normal human prostate cultures exhibited extensive cell-communication which was completely absent in all the examined tumor cells. This disrupted communication was associated with a decreased expression and an impaired posttranslational modification of Cx43 in these cells. Abundant immunostaining of gap junctional channels by a Cx43-antibody was observed in normal prostate cells but not in tumor cells. CONCLUSIONS: Our data provide further support for the hypothesis that loss of junctional communication is a critical step in progression to human prostate neoplasia.

Blotting, Western↗

PubMed1999

Reduced connexin43 expression in high-grade human brain glioma cells.

BACKGROUND AND OBJECTIVES: Connexin43 (cx43), a gap junction protein, is implicated in the suppression of tumor cell growth. Numerous cancer cells show a reduction or loss of cx43 expression compared to their normal counterparts. Our previous studies suggest that cx43 expression is decreased in a variety of human brain tumor cell lines. To further investigate the role of cx43 in the development of human gliomas, we performed the present study on human glioma grades I-IV. METHODS: Immunohistochemistry was performed on paraffin-embedded tissue sections of 18 human gliomas to analyze the expression levels of cx43 in different stages of human gliomas. RESULTS: High levels of cx43 were observed in all normal brain tissue and in glioma grades I and II. In contrast, the expression of cx43 was very weak in grade III gliomas and almost undetectable in grade IV gliomas. CONCLUSIONS: Our data support the hypothesis that reduction of cx43 is involved in the progression of human gliomas.

Astrocytoma↗

PubMed1999

Tumor promotion by hydrogen peroxide in rat liver epithelial cells.

Reactive oxygen species, including H2O2, play an important role in the tumor promotion process. Using an in vitro model of tumor promotion involving the rat liver epithelial oval cell line T51B, the tumor promoting activity of H2O2 in N-methyl-N'-nitro-N-nitrosoguanidine-initiated cells was studied. In this assay system, the promoting effect of H2O2 is evidenced by the formation of colonies in soft agar, appearance of foci in monolayer culture, disruption of gap junction communication (GJC) in foci areas and growth at higher saturation densities. H2O2 preferentially induced the expression of c-fos, c-jun, c-myc and egr-1, while JunB and JunD levels remained almost unchanged. H2O2 also induced hyperphosphorylation of Cx43 and disruption of GJC. The effects of H2O2 on tumor promotion, induction of immediate early (IE) genes and disruption of GJC are blocked by antioxidants. These results suggest that H2O2 acts as a tumor promoter in rat liver non-neoplastic epithelial cells and that the induction of IE genes and disruption of GJC are two possible targets of H2O2 during the tumor promotion process.

Animals↗

PubMed1999

Changes in postural muscle dynamics as a function of age.

Histologic studies have demonstrated both a decrease in size and loss in number of type II muscle fibers with increasing age. Although these age-related histologic changes are believed to result in decreased strength and functional capacity, age-related changes in muscle force dynamics have not been clearly elucidated. Using vibromyographic (VMG) techniques, we recorded muscle activity of the soleus in 40 healthy adult volunteers spanning the age range of 20-82 years to test whether changes in postural muscle dynamics, in the frequency range of 0.1-50 Hz, were also associated with age. Although muscle dynamics below 15 Hz do not change with aging, the 30-50 Hz frequency components of the VMG were found to change significantly with advancing age (r = -.619, p = .0001). This was observed in both sexes independently. The observed age-related changes in muscle force dynamics demonstrate distinct physiologic alterations in muscle fiber activity. Further research will be required to fully elucidate the relationship between age-related changes in muscle fiber activity and other age-related conditions such as postural instability and osteoporosis.

Adult↗

PubMed1998

Reversion of the neoplastic phenotype of human glioblastoma cells by connexin 43 (cx43).

Connexins (cx), structural components of gap junction, are believed to play a role in the regulation of cell proliferation and suppression of the neoplastic phenotype. We used human brain glioblastoma tumor cells as a model system to test this hypothesis. Western blot and reverse transcription-PCR analysis indicate that the expression levels of the gap junction protein connexin 43 (cx43) are profoundly decreased in several human brain tumor cell lines examined. Transfection of human cx43 into human glioblastoma cell lines U251 and T98G profoundly reduces cell proliferation in monolayer culture, in soft agar, and in athymic nude mice. Surprisingly, these effects are not associated with the establishment of gap junction communication in cx43 transfected cells. We conclude that the loss of cx43 expression may play a role in the development of human gliomas and that cx43 acts as a tumor suppressor gene to human glioblastoma.

Animals↗

PubMed1998

Suppression of human fibrosarcoma cell growth by transcription factor, Egr-1, involves down-regulation of Bcl-2.

Previously, we showed that the transcription factor Egr-1 suppressed the proliferation of v-sis transformed NIH3T3 cells and also a number of human tumor cells. Here, we investigate the possible mechanisms responsible for this function. We show that transfected Egr-1 in human fibrosarcoma cells HT1080 leads to down-regulation of Bcl-2. Transient CAT transfection assays reveal that expression of Egr-1 suppresses Bcl-2 promoter activity in a dose-dependent manner. Furthermore, overexpression of Bcl-2 in Egr-1-expressing HT1080 cells enhanced cell proliferation in monolayer culture and increased anchorage-independent growth. Our results suggest that suppression of tumor cell proliferation by Egr-1 may be at least partially mediated through the down-regulation of Bcl-2.

Blotting, Western↗

PubMed1998

Expression of multiple apoptosis-regulatory genes in human breast cancer cell lines and primary tumors.

The expression of several apoptosis-regulating genes was evaluated in 9 human breast cancer cell lines, 2 immortalized human mammary epithelial lines, 1 normal breast tissue biopsy, and 3 primary breast tumors, using a multiple antigen detection (MAD) immunoblotting method. The anti-apoptotic proteins Bcl-2, Bcl-X(L), Mcl-1, and BAG-1 were present at immunodetectable levels in 7, 10, 10, and 9 of the 11 lines. Comparing these 11 cell lines among themselves revealed that steady-state levels of Bcl-2, Bcl-X(L), Mcl-1, and BAG-1 were present at relatively higher levels in 4, 6, 5, and 5 of the lines, respectively. In contrast, the pro-apoptotic proteins Bax and Bak were detected in all 11 cell lines, and were present at relatively higher levels in 10 and 5 of the 11 lines, respectively. The Interleukin-1beta converting enzyme (ICE) homolog CPP32 (Caspase-3) was expressed in 10/11 breast cell lines. High levels of p53 protein, indicative of mutant p53, were found in 8 of the 11 lines and correlated inversely with Bax expression (p = 0.01). Bcl-2 and BAG-1 protein levels were positively correlated (p = 0.03). Immunoblot analysis of primary adenocarcinomas revealed expression of the anti-apoptotic proteins Bcl-2, Bcl-X(L), Mcl-1, and BAG-1, as well as the pro-apoptotic proteins Bax, Bak, and CPP32, in at least 2 of the 3 tumors examined. Immunohistochemical analysis was also performed for all of these proteins using 20 paraffin-embedded breast cancer biopsy specimens that all contained residual normal mammary epithelium in combination with both invasive cancer and carcinoma in situ. All of these apoptosis-regulating proteins were detected in primary breast cancers, though the percentage of immunopositive tumor cells varied widely in some cases. Comparisons of the intensity of immunostaining in normal mammary epithelium and invasive carcinoma suggested that Bcl-2 immunointensity tends to be lower in cancers than normal breast epithelium (p = 0.03), whereas CPP32 immunointensity was generally higher in invasive cancers (p < 0.0001). Taken together, the results demonstrate expression of multiple apoptosis-modulating proteins in breast cancer cell lines and primary tumors, suggesting complexity in the regulation of apoptosis in these neoplasms of mammary epithelial origin.

Apoptosis↗

PubMed1998

Egr-1 inhibits apoptosis during the UV response: correlation of cell survival with Egr-1 phosphorylation.

UV irradiation of normal or immortalized cells induces a rapid increase in the expression of several transcription factors and is thought to serve a protective function. The human fibrosarcoma cell line, HT1080 clone H4, expresses almost undetectable levels of Egr-1 and does not respond to UV-C irradiation by the induction of Egr-1. The H4 cells are hypersensitive to UV which induces apoptosis and reduces clonogenicity. The introduction of exogenous Egr-1 into H4 (H4E9 and H4E4 cell-lines) confers protection from UV damage as measured by a number of assays. In both NIH3T3 (with inducible Egr-1) and H4E9 (constitutive Egr-1) cells, UV irradiation gave enhanced transactivation of Egr-1 reporters that correlated with phosphorylated Egr-1. Studies using inhibitors indicated that protein kinase-C and tyrosine kinases are involved in the anti-apoptotic effects of Egr-1 after UV damage. This is the first description of a biological effect of phosphorylated Egr-1.

3T3 Cells↗

PubMed1997

Reciprocal modulation between Sp1 and Egr-1.

Many ubiquitously expressed genes, including oncogenes, lack a proximal TATA or CAAT box but have a region of G + C-rich sequences that appears to replace the usual promoter initiation site. The zinc-finger protein Sp1 is one of the prevalent activators of these genes. The Egr-1 zinc-finger protein has a similar binding site and if the two sites occur in the same region, a variety of activation or inhibitory responses may be obtained. We show that competition between the two factors for overlapping sites on growth-promoting genes could explain why the overexpression of Egr-1 suppresses transformed growth in a number of cell types [Huang et al. (1995): Cancer Res 55:5054-5062; Huang et al. (1997): Int J Cancer]. We demonstrate here that Egr-1 and Sp1 can bind to the same G + C-rich sites and that Egr-1 can displace Sp1 and hence inhibit its activity. We measured the responses of synthetic consensus binding sites and natural promoter sequences linked to a reporter gene and showed that Egr-1 inhibited the activation of transcription by Sp1 on overlapping Sp1/Egr-1 sites. In contrast, Sp1 activity could be augmented by Egr-1 at nonoverlapping sites in the Egr-1 gene promoter, in transient reporter gene studies in Drosophila SL2 cells. In addition, over-expression of exogenous Sp1 in mammalian cells, also leads to increased Egr-1 protein expression, which further inhibits Sp1 transactivation of numerous genes. Therefore, we can account for some of the complex responses of G + C-rich enhancer/promoters by a form of "facilitated inhibition" of Sp1 by Egr-1 at overlapping sites.

3T3 Cells↗