Biomedical subjects

Hong Song

Publications and source records attributed to Hong Song.

29 records · Page 2Linked to original sources

PubMed2002

Aggregation kinetics of well and poorly differentiated human prostate cancer cells.

Aggregation of attachment-dependent animal cells represents a series of motility, collision, and adhesion events applicable to such diverse fields as tissue engineering, bioseparations, and drug testing. Aggregation of human prostate cancer cells in liquid-overlay culture was modeled using Smoluchowski's collision theory. Using well (LNCaP) and poorly differentiated (DU 145 and PC 3) cell lines, the biological relevance of the model was assessed by comparing aggregation rates with diffusive and adhesive properties. Diffusion coefficients ranged from 5 to 90 microm(2)/min for single LNCaP and PC 3 cells, respectively. Similar diffusivities were predicted by the persistent random walk model and Einstein relation, indicating random motion. LNCaP cells were the most adhesive in our study with reduced cell shedding, 100% adhesion probability, and enhanced expression of E-cadherin. There was an increase in DU 145 cells staining positive for E-cadherin from nearly 20% of single cells to uniform staining across the surface of all aggregates; under 30% of PC 3 aggregates stained positive. Aggregation rates were more consistent with adhesive properties than with motilities, suggesting that aggregation in our study was reaction-controlled. Relative to other assays employed here, aggregation rates were more sensitive to phenotypic differences in cell lines and described size-dependent changes in aggregation at a finer resolution. In particular, model results suggest similar aggregation rates for two-dimensional DU 145 and PC 3 aggregates and upwards of 4-fold higher rates for larger three-dimensional DU 145 spheroids, consistent with expression of E-cadherin. The kinetic model has application to spheroid production, to cell flocculation and as an adhesion assay.

Cell Adhesion↗

PubMed2002

Na+ pump alpha 2-subunit expression modulates Ca2+ signaling.

The role of the Na(+) pump alpha(2)-subunit in Ca(2+) signaling was examined in primary cultured astrocytes from wild-type (alpha(2)+/+ = WT) mouse fetuses and those with a null mutation in one [alpha(2)+/- = heterozygote (Het)] or both [alpha(2)-/- = knockout (KO)] alpha(2) genes. Na(+) pump catalytic (alpha) subunit expression was measured by immunoblot; cytosol [Na(+)] ([Na(+)](cyt)) and [Ca(2+)] ([Ca(2+)](cyt)) were measured with sodium-binding benzofuran isophthalate and fura 2 by using digital imaging. Astrocytes express Na(+) pumps with both alpha(1)- ( approximately 80% of total alpha) and alpha(2)- ( approximately 20% of total alpha) subunits. Het astrocytes express approximately 50% of normal alpha(2); those from KO express none. Expression of alpha(1) is normal in both Het and KO cells. Resting [Na(+)](cyt) = 6.5 mM in WT, 6.8 mM in Het (P > 0.05 vs. WT), and 8.0 mM in KO cells (P < 0.001); 500 nM ouabain (inhibits only alpha(2)) equalized [Na(+)](cyt) at 8 mM in all three cell types. Resting [Ca(2+)](cyt) = 132 nM in WT, 162 nM in Het, and 196 nM in KO cells (both P < 0.001 vs. WT). Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) Ca(2+) pumps and unloads the ER, induces transient (in Ca(2+)-free media) or sustained (in Ca(2+)-replete media) elevation of [Ca(2+)](cyt). These Ca(2+) responses to 10 microM CPA were augmented in Het as well as KO cells. When CPA was applied in Ca(2+)-free media, the reintroduction of Ca(2+) induced significantly larger transient rises in [Ca(2+)](cyt) (due to Ca(2+) entry through store-operated channels) in Het and KO cells than in WT cells. These results correlate with published evidence that alpha(2) Na(+) pumps and Na(+)/Ca(2+) exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of alpha(2) Na(+) pump activity can elevate local [Na(+)] and, via Na(+)/Ca(2+) exchange, [Ca(2+)] in the tiny volume of cytosol between the plasma membrane and ER. This, in turn, augments adjacent ER Ca(2+) stores and thereby amplifies Ca(2+) signaling without elevating bulk [Na(+)](cyt).

Animals↗

PubMed2002

Inhibition of cytolytic T lymphocyte proliferation by autologous CD4+/CD25+ regulatory T cells in a colorectal carcinoma patient is mediated by transforming growth factor-beta.

Cancer patients often develop CTLs that lyse autologous tumor cells in culture. However, tumors can progress in vivo despite the presence of CTLs. Various mechanisms have been reported to down-modulate CTL functions. In this study, the role of CD4+/CD25+ regulatory T cells in CTL induction and proliferation of established CTLs was investigated in a patient with CRC. CD4+ cytotoxic and regulatory T-cell lines were derived from the peripheral blood mononuclear cells of the same patient in mixed-lymphocyte tumor culture. The cytotoxic T-cell line and a clonal derivative specifically lysed the autologous tumor cells but not the B lymphocytes. Only HLA-A1-matched allogeneic CRC cells were lysed by the CTL clone. The clone produced IFN-gamma and TNF-alpha. The regulatory CD4+/CD25+ T-cell line was tumor cell-dependent in its growth but did not lyse autologous tumor cells. This T-cell line suppressed pokeweed mitogen responses of allogeneic lymphocytes, proliferative activity of the established, autologous CTLs, and induction of CTLs in autologous, freshly isolated peripheral blood mononuclear cells. The immunosuppressive effect of the CD4+/CD25+ regulatory T cells was mediated by transforming growth factor-beta and did not require cell-to-cell contact. Thus, although CRC patients can develop specific CTLs against their tumors, the development of regulatory T cells may allow the escape of tumor cells from immune surveillance by the CTLs in vivo.

Animals↗

PubMed2002

Inhibition of tumor growth by recombinant vaccinia virus expressing GA733/CO17-1A/EpCAM/KSA/KS1-4 antigen in mice.

The human colorectal carcinoma (CRC)-associated GA733 antigen (Ag), also named CO17-1A/EpCAM/KSA/KS1-4, has been a useful target in passive immunotherapy of CRC patients with monoclonal antibody (mAb) and in active immunotherapy with anti-idiotypic antibodies or with recombinant protein. These approaches have targeted single epitopes (monoclonal anti-GA733 antibodies and anti-idiotypic antibodies) or extracellular domain epitopes (recombinant protein), primarily by B cells. To determine whether a reagent that induces immunity to a larger number of both B- and T-cell epitopes might represent a superior vaccine, we analyzed the capacity of full-length GA733 Ag expressing multiple potentially immunogenic epitopes and encoded by recombinant vaccinia virus (VV GA733-2) to induce humoral, cellular, and/or protective immunity in mice. VV GA733-2 induced Ag-specific antibodies that reacted predominantly to unknown epitopes on the Ag and lysed Ag-positive CRC targets in conjunction with murine peritoneal macrophages as effector cells. Immunized mice developed Ag-specific, proliferative and delayed-type hypersensitive lymphocytes. VV GA733-2 inhibited growth of ras-transformed syngeneic tumor cells expressing the human GA733 Ag in mice. These results suggest the potential of VV GA733-2 as a candidate vaccine for patients with CRC, possibly in combination with recombinant GA733-2-expressing adenovirus, which has been shown to induce cytolytic antibodies and T cells as well as tumor protective effects in mice. The combined vaccine approach may be superior to the use of either vaccine alone in patients who are pre-immune to both viruses.

3T3 Cells↗

PubMed2002

Differentiation kinetics of in vitro 3T3-L1 preadipocyte cultures.

Engineering autologous adipose constructs from cell culture is a promising strategy to overcome limitations of conventional soft-tissue implants. A methodology is presented to experimentally determine and mathematically model the differentiation kinetics of in vitro 3T3-L1 preadipocyte cultures that can aid in construct design. Relative rates of morphological and interfacial events during adipogenesis were compared. Model results suggest that maturation of an intermediate multilocular phenotype was the rate-limiting step in morphological differentiation and had an intrinsic rate of 0.012 day(-1). Dislodgment of multilocular fat cells was the primary mechanism of cell loss during adipogenesis. The maximum rate of lipid droplet nucleation was predicted to precede that of coalescence by 10 days and to be three times faster. Coalescence probability was estimated to decrease from 33 to 11% for 4- and 8-microm-diameter droplets, respectively. Fluid drainage and the cytoskeleton between droplets could have impeded coalescence. The kinetic analysis suggests that droplet ripening was the dominant mechanism of lipid production. Applications of this research include engineering of an adipose construct and predicting surgical outcome of patients requiring soft-tissue augmentation.

3T3-L1 Cells↗

PubMed2002

Organization of Ca2+ stores in vascular smooth muscle: functional implications.

Much evidence suggests that caffeine/ryanodine (Caf/Ry)-releasable and inositol-1,4,5-trisphosphate (InsP3)-releasable Ca2+ stores in the sarcoplasmic reticulum (SR) of smooth muscles are at least partially distinct. We directly visualized SR stores in primary-cultured rat mesenteric artery myocytes with high-resolution digital imaging and the low-affinity Ca2, indicator, Furaptra (Kd = 75.6 microM). The SR appears to be a continuous tubular network. Nevertheless, SR Ca2+ stores are organized into small, separate, functionally independent compartments. Cyclopiazonic acid (CPA; inhibits SR (Ca2+ pump) and Caf (or Ry) release Ca2+ from different, spatially distinct compartments. Similar heterogeneity is seen with serotonin (acts via InsP3), which unloads only the CPA-sensitive compartments. Some of the SR ('junctional' SR; jSR) lies within 12-15 nm of the plasmalemma (PL). The jSR, the overlying PL microdomains, and the intervening, tiny volume of cytosol form junctional complexes ('PLasmERosomes'). Na+ pumps with high-ouabain-affinity alpha2 or alpha3 subunits, Na+/Ca2+ exchangers, and store-operated channels are confined to these PL microdomains, whereas Na+ pumps with low-ouabain-affinity alpha1 subunits and plasma membrane Ca2+ pumps are uniformly distributed. As a result of this organization, low-dose ouabain can selectively modulate Na+ and Ca2+ concentrations in the PLasmERosomes and jSR Ca2+ stores, and can thereby regulate Ca2+ signalling.

Adenosine Triphosphatases↗

PubMed2002

[Effects of Helicobacter pylori infection on porcine gastric epithelial cell proliferation].

OBJECTIVE: To investigate the association between porcine gastric epithelial cell proliferation and Helicobacter pylori (Hp) infection. METHOD: Animal models of gastritis associated with Hp infection were established using "Chinese No.1 pigs". The expressions of proliferating cell nuclear antigen (PCNA) and DNA polyploid content in porcine gastric epithelial cells were quantitatively assayed by means of immunohistochemistry and Feulgen stain respectively. RESULTS: The values of PCNA labeling index (LI) in the porcine gastric epithelial cells of the experimental group were significantly higher than those of the control group (40.95+/-3.60 vs 29.4+/-12.82, P<0.01). The DNA diploid or approximate diploid content in the gastric mucosa of the pigs was significantly lower (70.78% vs 90.65%, P<0.01), but the proliferation polyploid and non-doubleploid of DNA significantly higher in the experimental group than in the control group (P<0.01). CONCLUSION: Hp infection may play a role to enhance the proliferation of gastric epithelial cells in pigs.

Animals↗

PubMedDate not supplied

Monte Carlo simulation of LNCaP human prostate cancer cell aggregation in liquid-overlay culture.

Neoplastic cells self-assemble in liquid-overlay cultures into multicellular spheroids that resemble micrometastases and avascular regions of larger tumors. A Monte Carlo simulation based on Meakin's cluster-cluster aggregation model resolved the physical mechanisms by which LNCaP human prostate cancer cells aggregate in this environment. The best-fit solution suggests that LNCaP cells aggregate with an adhesion probability of 0.5% when they migrate within a radius of influence between cell centers of 180 microm, 10 times the cell diameter. The sweeping radius of influence is indicative of cell tethering and/or chemotaxis and results in an intrinsic rate of self-aggregation that increases from k(11) = 1.5 h(-1) for single cells to k(1010) = 17.5 h(-1) for 10-mers. Similar rates are predicted by Smoluchowski's collision theory (1), suggesting that they are inherent properties of LNCaP liquid-overlay culture. Aggregates form more compact structures in culture than during simulation as measured by the fractal dimension: D(F) = 1.74 +/- 0.04 for 10-mers in culture vs D(F) = 1.25 +/- 0.10 for simulated 10-mers. Additional restructuring would further extend the radius of influence and diminish adhesion. Applications of this work include the production of highly viable spheroids for drug testing and basic oncological research.

Cell Adhesion↗

PubMedDate not supplied

Spatial composition of prostate cancer spheroids in mixed and static cultures.

Aggregation of neoplastic cells produces multicellular spheroids resembling micrometastases. The objective of this study was to investigate the effects of mixing culture medium on the spatial composition of spheroids prepared from well (LNCaP) and poorly (DU 145) differentiated human prostate cancer cells. Spheroids were cultured in a mixed suspension within a high-aspect rotating wall vessel and static liquid-overlay plate. Results from this study demonstrate that mixed cultures consistently manifested differences in morphology and composition between DU 145 and LNCaP spheroids. For example, 40 +/- 12% of DU 145 cells were Ki-67 positive 100 microm from the surface within mixed spheroids versus 0% for LNCaP cells; there was no significant difference in this spatial profile for static cultures. The results suggest that poorly differentiated spheroids may be more likely to experience a change in composition from mixing culture medium than well-differentiated spheroids, due to low tissue density. Immunostaining for P-glycoprotein is representative of this trend; average staining intensity increased 50% for DU 145 spheroids on mixing but was unchanged for LNCaP spheroids. The effects of mixing on spheroid composition were attributed to faster interstitial mass transport. Applications include drug development and delivery, as well as basic research on drug action and resistance.

Cell Line, Tumor↗

PubMedDate not supplied

Immunohistochemical analysis of differentiation in static and mixed prostate cancer spheroids.

Neoplastic multicellular spheroids are in vitro models of solid tumors employed in drug testing and basic research. This study compares differentiation in static and mixed prostate cancer spheroids. Staining intensity of prostate specific antigen (PSA) was down-regulated upon mixing, from 0.21 +/- 0.03 to 0.13 +/- 0.03 in LNCaP multicellular spheroids, and from 0.13 +/- 0.04 to 0.03 +/- 0.02 in DU 145 multicellular spheroids. This was accompanied by 65% increase in the expression of cytokeratins 8 and 18 in DU 145 spheroids. PSA expression extended 60 micro m within static spheroids and was disrupted in mixed culture. Diminished PSA expression and spatial organization suggests a more aggressive cancer. Higher cytokeratin expression could result from either differentiation towards a luminal phenotype or activation of the Ras pathway during dedifferentiation. Thus, the existing paradigm of differentiation established for normal tissue does not apply for our neoplastic spheroids. Cell dedifferentiation is attributed to improved interstitial transport and synthesis of extracellular matrix.

Animals↗

PubMedDate not supplied

Restructuring dynamics of DU 145 and LNCaP prostate cancer spheroids.

Neoplastic cells acquire multidrug resistance as they assemble into multicellular spheroids. Image analysis and Monte Carlo simulation provided an insight into the adhesion and motility events during spheroid restructuring in liquid-overlay culture of DU 145 and LNCaP human prostate cancer cells. Irregularly shaped, two-dimensional aggregates restructured through incremental cell movements into three-dimensional spheroids. Of the two cultures examined, restructuring was more pronounced for DU 145 aggregates. Motile DU 145 cells formed spheroids with a minimum cell overlay of 30% for 25-mers as estimated by simulation versus 5% for adhesive LNCaP cells in aggregates of the same size. Over 72 h, the texture ratio increased from 0.55 +/- 0.05 for DU 145 aggregates with projected areas exceeding 2000 microm2 to a value approaching 0.75 +/- 0.02 (P < 0.05). For LNCaP aggregates of comparable size, the increase in texture ratio was more modest, less than 15% during the same time period (P < 0.05). Combined, these data suggest that motility events govern the overall rate of spheroid restructuring. This information has application to the chemosensitization of solid tumors and kinetic modeling of spheroid production.

Cell Aggregation↗