Biomedical subjects

C J Fong

Publications and source records attributed to C J Fong.

12 recordsLinked to original sources

PubMed2006

Effects of culture conditions on estrogen-mediated hepatic in vitro gene expression and correlation to in vivo responses.

Refinement of in vitro systems for predictive toxicology is important in order to develop high-throughput early toxicity screening assays and to minimize animal testing studies. This study assesses the ability of mouse Hepa-1c1c7 hepatoma cell model under differing culture conditions to predict in vivo estrogen-induced hepatic gene expression changes. Custom mouse cDNA microarrays were used to compare Hepa-1c1c7 temporal gene expression profiles treated with 10 nM 17beta-estradiol (E2) in serum-free and charcoal-stripped serum supplemented media at 1, 2, 4, 8, 12, and 24 h. Stripped serum supplemented media increased the number gene expression changes and overall responsiveness likely due to the presence of serum factors supporting proliferation and mitochondrial activity. Data from both experiments were compared to a gene expression time course study examining the hepatic effects of 100 microg/kg 17alpha-ethynyl estradiol (EE) in C57BL/6 mice at 2, 4, 8, 12, 18, and 24 h. Only 18 genes overlapped between the serum-free and in vivo studies, whereas 238 genes were in common between Hepa-1c1c7 cells in stripped serum data and C57BL/6 liver samples. Stripped serum cultured cells exhibited E2-elicited gene expression changes associated with proliferation, cytoskeletal re-organization, cholesterol uptake and synthesis, increased fatty acid beta-oxidation, and oxidative stress, which correlated with in vivo hepatic responses. These results demonstrate that E2 treatment of Hepa-1c1c7 cells in serum supplemented media modulate responses in selected pathways which appropriately model estrogen-elicited in vivo hepatic responses.

Animals↗

PubMed2005

Protocols for the assurance of microarray data quality and process control.

Microarrays represent a powerful technology that provides the ability to simultaneously measure the expression of thousands of genes. However, it is a multi-step process with numerous potential sources of variation that can compromise data analysis and interpretation if left uncontrolled, necessitating the development of quality control protocols to ensure assay consistency and high-quality data. In response to emerging standards, such as the minimum information about a microarray experiment standard, tools are required to ascertain the quality and reproducibility of results within and across studies. To this end, an intralaboratory quality control protocol for two color, spotted microarrays was developed using cDNA microarrays from in vivo and in vitro dose-response and time-course studies. The protocol combines: (i) diagnostic plots monitoring the degree of feature saturation, global feature and background intensities, and feature misalignments with (ii) plots monitoring the intensity distributions within arrays with (iii) a support vector machine (SVM) model. The protocol is applicable to any laboratory with sufficient datasets to establish historical high- and low-quality data.

Artificial Intelligence↗

PubMed2005

Comparative microarray analysis of basal gene expression in mouse Hepa-1c1c7 wild-type and mutant cell lines.

Hepa-1c1c7 wild-type and benzo[a]pyrene-resistant derived mutant cell lines have been used to elucidate pathways and mechanisms involving the aryl hydrocarbon receptor (AhR). However, there has been little focus on other biological processes which may differ between the isolated lines. In this study, mouse cDNA microarrays representing 4858 genes were used to examine differences in basal gene expression between mouse Hepa-1c1c7 wild-type and c1 (truncated Cyp1a1 protein), c4 (AhR nuclear translocator, ARNT, deficient), and c12 (low AhR levels) mutant cell lines. Surprisingly, c1 mutants exhibited the greatest number of gene expression changes compared to wild-type cells, followed by c4 and c12 lines, respectively. Differences in basal gene expression were consistent with cell line specific variations in morphology, mitochondrial activity, and proliferation rate. MTT and direct cell count assays indicate both c4 and c12 mutants exhibit increased proliferative activity when compared to wild-type cells, while the c1 mutants exhibited decreased activity. This study further characterizes Hepa-1c1c7 wild-type and mutant cells and identifies significant differences in biological processes that should be considered when conducting comparative mechanistic studies with these lines.

Animals↗

PubMed2002

Ability of structurally diverse natural products and synthetic chemicals to induce gene expression mediated by estrogen receptors from various species.

The ability of 14 structurally diverse estrogenic compounds to induce reporter gene expression mediated by estrogen receptors (ERs) from different species was examined. MCF-7 cells were transiently transfected with a Gal4-regulated luciferase reporter gene (17m5-G-Luc) and Gal4-ER chimeric receptors containing the D, E and F domains of the human alpha (Gal4-hERalphadef), mouse alpha (Gal4-mERalphadef), mouse beta (Gal4-mERbetadef), chicken (Gal4-cERalphadef), green anole (Gal4-aERalphadef), Xenopus (Gal4-xERdef) or rainbow trout alpha ERs (Gal4-rtERalphadef). The efficacy of 17beta-estradiol (E2) in inducing reporter gene expression was similar among the different constructs overall, with EC(50) values ranging from 0.05 to 0.7nM. However, Gal4-rtERalphadef had an EC(50) value at 37 degrees C of 28nM, though at 20 degrees C an EC(50) value of 1nM was observed. Despite a similar response to E2 treatment among the ERs, many differences were observed in the magnitude of the response to other structurally diverse chemicals. For example, coumestrol induced Gal4-mERbetadef- and Gal4-aERdef-mediated reporter gene expression 164- and 8-fold greater, respectively, than mediated with the other Gal4-ERs. As well, in contrast to results with other Gal4-ERs, alpha-zearalenol consistently induced Gal4-rtERalphadef-mediated reporter gene activity at lower concentrations than did E2. Overall, the results demonstrate that selected estrogenic compounds exhibit a differential ability to induce reporter gene activity mediated by ERs from different vertebrate species. These data also highlight the importance of incubation temperature when examining rtERalpha-mediated activity.

Animals↗

PubMed2001

Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones.

This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62. 2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Isolation of individual colonies from these stocks demonstrated that 7.1% of the original 1189 stocks contained both a correct and an incorrect plasmid. 5.9% of the original 1189 stocks contained multiple, distinct, incorrect plasmids, indicating the likelihood of multiple contaminating events. While only 739 of the stocks purchased contained the desired cDNA clone, agarose gel pre-screening, colony isolation and similarity searching of dbEST allowed for the identification of an additional 420 clones that would have otherwise been discarded. Considering the high error rate in this subset of the IMAGE cDNA clone set, the use of sequence verified clones for cDNA microarray construction is warranted. When this is not possible, pre-screening non-sequence verified clones with agarose gel electrophoresis provides an inexpensive and efficient method to eliminate contaminated clones from the probe set.

Animals↗

PubMed1993

Effect of retinoic acid on the proliferation and secretory activity of androgen-responsive prostatic carcinoma cells.

We studied the effect of retinoic acid on the growth and secretory activity of the androgen-responsive prostatic carcinoma cell line LNCaP. Our data showed that retinoic acid at 0.01 microM. stimulated the proliferation of LNCaP cells but inhibited their growth at 0.1 microM. under androgen-free conditions. In the presence of 0.1 nM. dihydrotestosterone (DHT), LNCaP cell proliferation was inhibited by 10 microM. retinoic acid but not by lower concentrations of retinoic acid. Retinoic acid reduced LNCaP cell growth at concentrations of 0.1 microM. in the presence of 10 nM. DHT. Retinoic acid (10 microM.) also reduced the growth response of LNCaP cells to epidermal growth factor and transforming growth factor alpha and potentiated the inhibitory effect of transforming growth factor beta. In additional studies, retinoic acid induced a dose-dependent increase in prostate specific antigen (PSA) secretion at concentrations of 0.1 to 1 microM. Dihydrotestosterone (10 nM.) also enhanced the secretion of PSA by LNCaP cells, and this effect was potentiated in a dose-dependent fashion by the addition of retinoic acid at 0.1-10 microM. Competitive binding studies showed that retinoic acid did not bind to androgen receptors. Overall, retinoic acid had a biphasic effect on LNCaP proliferation and promoted the secretion of PSA. The biphasic effect of retinoic acid on LNCaP growth should be considered in designing in vivo studies to determine the impact of retinoic acid on solid prostatic tumor growth. In addition, the ability of retinoic acid to increase PSA secretion may complicate the interpretation of serum PSA levels used for diagnostic and prognostic purposes.

Androgens↗

PubMed1992

Epidermal growth factor receptor monoclonal antibody inhibits constitutive receptor phosphorylation, reduces autonomous growth, and sensitizes androgen-independent prostatic carcinoma cells to tumor necrosis factor alpha.

Results of recent studies indicate that cultured, androgen-independent prostatic carcinoma cells synthesize and secrete transforming growth factor alpha, which interacts with epidermal growth factor receptors (EGFRs) to promote autonomous growth. In the present study, we evaluated the expression and constitutive activation of EGFRs in normal prostatic epithelial cells and the androgen-independent prostatic carcinoma cell lines PC3 and DU145. Our studies showed that cultured normal epithelial cells and androgen-independent prostatic carcinoma cells actively synthesize and exhibit constitutive phosphorylation of the M(r) 170,000 EGFR. The addition of monoclonal anti-EGFR reduced receptor phosphorylation and significantly inhibited the proliferation of prostatic tumor cells. The observed reduction in EGFR phosphorylation could be partially attributed to an antibody-induced decrease in the expression of metabolically labeled EGFR. Results of further studies showed that anti-EGFR enhanced the sensitivity of PC3 cells to the cytotoxic and cytostatic effects of tumor necrosis factor alpha. These studies demonstrate that constitutive activation of EGFR in androgen-independent prostatic carcinoma plays a functional role in the regulation of cellular proliferation in vitro. In addition, the enhanced sensitivity of prostatic carcinoma cells to tumor necrosis factor alpha in the presence of anti-EGFR provides a rationale for the further investigation of combination therapy in the treatment of disseminated, androgen-independent disease.

Antibodies, Monoclonal↗

PubMed1992

Regulation of prostatic carcinoma cell proliferation and secretory activity by extracellular matrix and stromal secretions.

Previous studies from our laboratory have shown that reconstituted basement membrane and stromal secretory products are important regulators of benign prostatic epithelial cell growth and differentiation. In the present study we evaluated the impact of extracellular matrix (ECM) and soluble stromal secretory products on the proliferation and secretory activity of the androgen-responsive prostatic carcinoma cell line LNCaP. In these studies, dihydrotestosterone (DHT) was a potent mitogen for LNCaP cells cultured on plastic or on type I collagen. The growth response to DHT was greatly attenuated when LNCaP cells were grown on prostatic stromal ECM. Cells grown on stromal ECM also exhibited clustered morphology compared to the monolayer growth observed on plastic and secreted elevated levels of prostate specific antigen (PSA) and prostatic acid phosphatase (PAP). These findings indicate that cultivation of LNCaP on stromal ECM will promote the expression of differentiated functions. In additional studies, stromal cell conditioned medium (SCM) significantly increased PSA/PAP secretion by LNCaP cells in the presence of 10 nM DHT. The enhancement of DHT-induced PSA/PAP secretion by SCM was most pronounced when LNCaP cells were grown on stromal ECM. SCM did not significantly alter LNCaP proliferation. These studies indicate that prostatic stromal ECM and soluble secretory products will promote differentiated function in cultured LNCaP cells. In addition, we show that DHT can act as either a growth or differentiation-promoting stimulus depending on the presence of stromal factors.

Acid Phosphatase↗

PubMed1992

Interaction of epidermal growth factor and transforming growth factor beta in human prostatic epithelial cells in culture.

The present study was conducted to study the interaction between epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in benign human prostatic epithelial cells in culture. Primary cultures of human prostatic epithelial cells were grown in complete WAJC, which consisted of WAJC-404 medium and, in addition to other defined additives, EGF and bovine pituitary extract (BPE). Incomplete WAJC contained the same composition except EGF and BPE were deleted. TGF-beta was added into media at concentrations of 0, 0.1, and 1.0 ng/ml. When cells were grown in complete WAJC, they proliferated rapidly. Cell proliferation was greatly suppressed when incomplete WAJC was used. Addition of TGF-beta to these cultures caused a significant reduction in the final cell number when either complete WAJC or incomplete WAJC was used. In additional experiments, cells were prelabeled with 3H-thymidine for 72 hr prior to treatment with TGF-beta. The percentage of radioactivity released into the medium at the end of a 6-day culture was used as an indication of the extent of cell death. Trypan blue exclusion test was also used to assess the extent of cell death. Addition of TGF-beta into complete WAJC did not significantly affect the extent of cell death beyond what was considered as the result of normal cellular turnover. Addition of TGF-beta into incomplete WAJC, however, caused a significant increase in the percent of cell death in the culture. These results demonstrated an interaction between EGF and TGF-beta in proliferation and cell death in human prostatic epithelia in culture. In the presence of EGF alone in the culture medium, prostatic epithelial cells were stimulated to proliferate. The rate of proliferation was greatly diminished when EGF was deleted from the medium or when TGF-beta was added in the presence of EGF. Finally, cell death was induced when TGF-beta was added into the medium in the absence of EGF.

Analysis of Variance↗

PubMed1992

Basic fibroblast growth factor: a potential mediator of stromal growth in the human prostate.

Studies were undertaken, using isolated prostatic epithelial and stromal cells, to evaluate the role of basic fibroblast growth factor (bFGF) in the regulation of benign prostatic growth. bFGF was detected in lysates, but not the conditioned media, of cultured prostatic epithelial and stromal cells by Western immunoblotting and immunoprecipitation of metabolically labeled proteins. Immunofluorescence analysis of benign human prostate localized the majority of bFGF to the prostatic stroma. In addition, bFGF was a potent stimulator of stromal cell proliferation in vitro, but was not mitogenic to cultured epithelial cells. Further studies demonstrated bFGF receptors (Kd = 258 pM; 61,400 receptors/cell) on stromal cells, but not epithelial cells. Epithelial cell-conditioned medium was mitogenic for stromal cells, suggesting the presence of paracrine interactions. However, bFGF does not appear to be the mediator of this interaction, since the mitogenic effect of epithelial cell-conditioned medium on stromal cells was not significantly reduced by the addition of anti-bFGF. Additional studies showed that concentrated stromal cell-conditioned medium was not mitogenic to cultured stromal cells under serum-free defined conditions, indicating the lack of an external autocine mechanism. These studies demonstrate that bFGF is actively synthesized by isolated prostatic epithelial and stromal cells, but is largely not secreted. Prostatic stroma, but not epithelia, are responsive to the mitogenic effect of bFGF in vitro. However, because of the limited secretion of bFGF by prostatic cells, the mechanism(s) of bFGF-mediated regulation of stromal growth remains unclear.

Cell Division↗

PubMed1992

Utilization of the Boyden chamber to further characterize in vitro migration and invasion of benign and malignant human prostatic epithelial cells.

In the present study, we assessed various tissue culture conditions on the motility and invasiveness of benign and malignant prostatic epithelial cells using the Boyden chamber system. In DME and RPMI-1640 media, benign human prostatic epithelial cells were unable to penetrate through a basement-membrane-coated filter. However, the prostatic carcinoma cell lines, PC3 and DU145, showed a significant frequency of invasion under those conditions. When cultured in the low-calcium WAJC 404 medium, benign prostatic epithelial cells efficiently penetrated through the basement-membrane-coated filter. PC3 and DU145 cells showed an attenuated capability to invade when cultured in WAJC 404 medium. The effect of extracellular calcium on the behavior of these cells in the Boyden chamber was further evaluated by gradual addition of calcium chloride in WAJC 404 medium. These studies showed that the number of benign prostatic epithelial cells penetrating through the filter decreased as the calcium ion concentration increased. Conversely, the number of PC3 cells invading through the filter increased as calcium ion concentration was increased to 0.4 mM and decreased at higher calcium ion concentrations. These results suggest that the extracellular calcium concentration is one of the factors which may affect cell behavior in the Boyden chamber system.

Basement Membrane↗

PubMed1991

Reconstituted basement membrane promotes morphological and functional differentiation of primary human prostatic epithelial cells.

Prostatic epithelial cells undergo rapid proliferation and lose their ability to synthesize and secrete prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) under standard tissue culture conditions. Herein, we compared the morphology, growth, secretory activity, and intermediate filament expression of human prostatic epithelial cells cultured on either standard tissue culture plastic or reconstituted basement membrane. Epithelial cells grown on plastic exhibited a 10-fold increase in proliferation and a higher percentage of cells in the S-phase of the cell cycle compared to cells cultured on basement membrane. However, cells grown on basement membrane secreted markedly higher levels of PSA and PAP. The basement membrane-induced enhancement of secretory activity was potentiated by dihydrotestosterone (DHT) and prostate stromal cell conditioned medium. Morphological studies showed that cells plated on basement membrane formed organoid-like clusters and maintained several aspects of differentiated epithelium including abundant secretory vesicles, microvilli, and desmosomes with associated cytoskeletal elements. Cultivation of epithelial cells on basement membrane components also suppressed the expression of vimentin, a mesenchymal intermediate filament polypeptide. However, cytokeratin expression was abnormal in cells grown on either surface. These results indicate that the differentiated properties of prostatic epithelial cells are promoted by cultivation on reconstituted basement membrane in the presence of DHT and stromal cell conditioned medium.

Acid Phosphatase↗