Possible role of cell cycle-dependent morphology, geometry, and mechanical properties in tumor cell metastasis.

Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells. As the cell cycle progressed at 37 degrees C, an increase in cell volume from 1400 microns 3 to 5700 microns 3 was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the "older" cells. Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system.     

A physical characterization of GAP A3 hybridoma cells: morphology, geometry, and mechanical properties

Morphological, geometrical, and rheological properties of the GAP A3 hybridoma cell line have been evaluated as a function of the cell cycle. Interference contrast video microscopy and scanning electron microscopy (SEM) showed that a sample of cells taken from the middle of the exponential growht phase displayed a range of cell morphologies, consistent with a heterogeneous growing culture. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (cortical tension and apparent cell viscosity) properties of single cells selected at random from a sample in the middle of the exponential growth phase. Consistent with the range of morphologies, cell volumes (1400 to 5700 mum(3)) and apparent viscosities (430 to 1.2 x 10(4) P) showed a wide range of values at 37 degrees C, demonstrating that a hybridoma cell line cannot be characterized by a single value for any one property, and that properties must be related to their cycle dependence when considering proliferating cells. Direct, video-microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle, allowed us to correlated distinct morphologies with phases of the cell cycle. As the cell cycle progresses, an increase in cell volume by a factor of 3 to 4is accompanied by an overall increase in apparent cell viscosity by approximately the same ratio, consistent with an accumulation of more cytoplasmic material in the older cells. Also, a decrease in average apparent viscosity by a factor of 10. These results are important in order to evaluate the possible role of certain structural, cell-cycle dependent features in shear and abrasion sensitivity. This is a problem of current concern in the bioreactor culture of mammalian cells.     

Tetrodotoxin suppresses morphological enhancement of the metastatic MAT-LyLu rat prostate cancer cell line

Voltage-gated Na⁺ channels are expressed by highly metastatic MAT-LyLu cells, but not by poorly metastatic AT-2 cells, derived from the rodent Dunning model of prostatic cancer. We have investigated the possible involvement of these channels in the morphological development of the cells. Incubation of both the MAT-LyLu and the AT-2 cell line for 24 h with the Na⁺ channel blocker tetrodotoxin (TTX) at 6 μM altered the morphology only of the MAT-LyLu cell line. TTX produced significant decreases in: (a) cell process length and (b) field diameter, and increases in (c) cell body diameter and (d) process thickness. Importantly, 6 μM TTX had no significant effects on proliferation rates or cellular toxicity. The results suggest that Na⁺ channel activity plays a significant role in determining the morphological development of MAT-LyLu cells in such a way as to enhance their metastatic potential. Received: 9 March 1998 / Accepted: 5 October 1998     

Effectiveness of vitamin A acetate for enhancing the production of lung cancer specific monoclonal antibodies

The antibody productivity of the human–human hybridoma cell line AE6, which produces the lung cancer specific human monoclonal antibody AE6F4, was enhanced fourfold upon stimulation with 1 μg/ml of vitamin A acetate for one day. The enhancement lasted for about two weeks, and could be repeated by another stimulation with vitamin A acetate. The enhancing effect of vitamin A acetate was influenced by the cell density. Enhancement was clearly observed when the cell density was under 10⁶ cells/ml. However, when the cell density was over 10⁷ cells/ml, enhancement was observed weakly or not at all. Although the enhancing effect of vitamin A acetate is not unique to AE6 cells, not all human–human hybridoma cell lines show increased productivity upon VA acetate stimulation. This study suggests that the response to vitamin A acetate may be related to the properties of a particular fusion partner which the hybridoma cell inherits. The efficacy of vitamin A acetate for production of human monoclonal antibodies using human–human hybridomas is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Active specific immunotherapy with hapten-modified autologous melanoma cell vaccine

 We have developed a novel approach to cancer immunotherapy – an autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP). This approach elicits significant inflammatory responses in metastatic sites and some objective tumor responses. Post-surgical adjuvant immunotherapy with DNP-modified melanoma vaccine in a setting of micrometastatic disease produces significant survival prolongation in stage III melanoma patients. Histologically, the inflammatory responses of the tumor consist of infiltration by lymphocytes, the majority of which are CD8⁺, HLA-DR⁺ T cells. T cells from these lesions tend to have mRNA for interferon γ. T cell receptor analysis suggests that the tumor-infiltrating T cells are clonally expanded. DNP-modified vaccine also induces T cells in the peripheral blood, which respond to DNP-modified autologous cells in a hapten-specific, MHC-restricted manner. Moreover, a T cell line generated from these lymphocytes responded to only a single HPLC fraction of MHC-associated, DNP-modified tumor peptides. Since inflammatory responses in metastases were not consistently associated with dramatic tumor regression, we considered the possibility of immunosuppression at the tumor site. We found that mRNA for the anti-inflammatory cytokine, interleukin-10 (IL-10) is expressed in most metastatic melanoma tissues and subsequently demonstrated that IL-10 protein is produced by melanoma cells. Thus the efficacy of DNP vaccine could be further enhanced by inhibition of IL-10 production or binding. Finally, we expect these results obtained with melanoma to be applicable to other human cancers. Received: 6 August 1996 / Accepted: 20 September 1996     

Multiple effects of mercury on cell volume regulation, plasma membrane permeability, and thiol content in the human intestinal cell line Caco-2

In a previous study, we characterized Cd–Hg interactions for uptake in human intestinal Caco-2 cells. We pursued our investigations on metal uptake from metal mixtures, focusing on the effects of Hg on cellular homeostasis. A 4-fold higher equilibrium accumulation value of 0.3 μmol/L ²⁰³Hg was measured in the presence of 100 μmol/L unlabeled Hg in the serum-free exposure medium without modification in the initial uptake rate. This phenomenon was eliminated at 4^∘C. Mercury induced an increase in tritiated water and [³H]mannitol uptakes for exposure times greater than 20 min. Incubations for 20 min and 30 min with 100 μmol/L Hg and 2 mmol/L N-ethylmaleimide (NEM) resulted in a 34% and 50% reductions in cellular thiol staining, respectively, with additive effects. Lactate dehydrogenase leakage and live/dead assays confirmed the maintenance of cell membrane integrity in Hg- or NEM-treated cells. We conclude that Hg may alter membrane permeability and increase cell volume without any loss in cell viability. This phenomenon is sensitive to temperature and could involve Hg interaction with membrane thiols, possibly related to solute transport. During metal uptake from metal mixtures, Hg may thus promote the uptake of other toxic metals by increasing cell volume and consequently cell capacity. Deceased 25 March 2004     

Rheology and Shear Stress of Centaurea calcitrapa Cell Suspension Cultures Grown in Bioreactor

Centaurea calcitrapa suspension cultures were grown either in Erlenmeyer flasks or in a mechanically stirred bioreactor. Its rheological behaviour, when fitted to the Oswald–de Waele model (power law), showed pseudoplastic characteristics in both cases. The flow behaviour index (n) decreased over the course of a growth cycle and the consistency index (K) increased, reached a value of 1.81 N sⁿ m⁻² run on 2 l bioreactor. Bioreactor cultivation of C. calcitrapa cells at different agitation rates (30, 60, 100 and 250 rpm), highlighted the influence of shear forces on cell viability loss (90–34%) and phenol accumulation (74–140 μg l⁻¹), due to increased stirring speeds. Analysis of these results suggests that this cell line is shear-sensitive. An empirical exponential correlation was defined between apparent viscosity and biomass concentration, under the studied conditions, giving the possibility to estimate the prevailing broth regime and to optimize bioreactor design. Revisions requested 10 October 2005; Revisions received 19 December 2005     

Cell suspension culture and plant regeneration in the latex-producing plant,Calotropis gigantea (Linn.) R. Br.

A procedure is outlined for the establishment of a proliferating cell suspension culture and subsequent plant regeneration of the latex-producing plant,Calotropis gigantea (Linn.) R. Br. Friable calluses were obtained by culturing hypocotyl explants on modified Murashige and Skoog medium with 2.69 μM α-naphthaleneacetic acid and 4.44 μM 6-benzyl-aminopurine. Friable calluses were transferred to modified Murashige and Skoog liquid medium containing 500 mg l⁻¹ casein hydrolysate, 5% (v/v) coconut water and 5% (w/v) sucrose to initiate suspension cultures. Suspensions were subcultured every 10–12 days and supplemented with 13.56 μM 2,4-dichlorophenoxyacetic acid (2,4-D). After 3–4 subcultures, suspensions consisted of small, highly cytoplasmic cell clumps and large vacuolate cells. Plating the suspension clumps on medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and culturing in darkness produced macroscopic calluses, which subsequently produced a high number of shoots when placed in light and supplemented with 2.22 μM 6-benzyl-aminopurine and 0.45 μM 2,4-dichlorophenoxyacetic acid. Shoots were rooted using Bonner's solution containing 0.49 μM indole-3-butyric acid, and the plants successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Single cell adhesion measuring apparatus (SCAMA): application to cancer cell lines of different metastatic potential and voltage-gated Na<Superscript>+</Superscript> channel expression

We have developed a simple yet effective apparatus, based upon negative pressure directed to the tip of a micro-pipette, to measure the adhesiveness of single cells. The “single cell adhesion measuring apparatus” (SCAMA) could differentiate between the adhesion of strongly versus weakly metastatic cancer cells as well as normal cells. Adhesion was quantified as “detachment negative pressure” (DNP) or “DNP relative to cell size” (DNPR) where a noticeable difference in cell size was apparent. Thus, for rat and human prostate and human breast cancer cell lines, adhesiveness (DNPR values) decreased in line with increased metastatic potential. Using the SCAMA, we investigated the effect of tetrodotoxin (TTX), a specific blocker of voltage-gated Na⁺ channels (VGSCs), on the adhesion of rat and human prostate cancer cell lines of markedly different metastatic potential. Following pretreatment with TTX (48 h with 1 μM), the adhesion values for the Mat-LyLu cells increased significantly 4.3-fold; there was no effect on the AT-2 cells. For the strongly metastatic PC-3M cells, TTX treatment caused a significant (∼30%) increase in adhesion. The adhesion of PNT2-C2 (“normal”) cells was not affected by the TTX pretreatment. The TTX-induced increase in the adhesiveness of the strongly metastatic cells was consistent with the functional VGSC expression in these cells and the proposed role of VGSC activity in metastatic cell behaviour. In conclusion, the SCAMA, which can be constructed easily and cheaply, offers a simple and effective method to characterise single-cell adhesion and its modulation.     

Encapsulated animal cell culture for the production of monoclonal antibody (MAb)

Biopolymer membrane was prepared using two oppositely charged natural biopolymer. The biopolymer membrane was used for the encapsulation of two hybridoma cell (ATCC CRL-1606, ATCC BH-8852) to produce monoclonal antibodies. In order to reduce the down stream steps, the pore size of the membrane was controlled to retain the monoclonal antibodies in the capsules based on the diffusion experiments with standard proteins. T-flask culture showed cell densities of 8×10⁷ cells/mL and 3×10⁷ cells/mL, and MAb concentrations of 506 μg/mL and 109 μg/mL for encapsulated ATCC CRL-1606 and HB-8852, respectively. Two liter perfusion culture with encapsulated ATCC HB-8852 was performed to enhance the MAb production. The MAb production of the encapsulated hybridoma increased considerably comparing to the culture using silicone tubing for oxygen transfer.     

Relationship between cell size, cell cycle and specific recombinant protein productivity

Centrifugal elutriation was used to produce cell cycle enrichedfractions of four commercially relevant recombinant cell lines,chosen to allow for variation in properties due to construct,expression system and parent cell type, from normally growingheterogeneous batch cultures. As these fractions had identicalculture histories and had not been subjected to any insult orstress which was likely to have adversely affected cellularmetabolism, they were ideal for further study of cellularproperties. Specific productivity, cell size and cell cyclestate of replicate elutriated fractions were measured for eachcell line. Results showed that cell size was the major cellulardeterminant of productivity for all cell lines examined. Productformation was not restricted to any particular cell cycle phaseand in all cases, production occurred irrespective of cell cyclephase. Specific productivity was lowest when the majority ofcells in the fraction were G₁, intermediate when themajority of cells in the fraction were S phase and greater whenthe majority of cells in the fraction were in G₂/M. However, the evidence suggests that size is the major cellulardeterminant of productivity; the apparent relationship betweencell cycle and productivity is secondary and can simply beascribed to the increasing size of cells as they progress thoughthe cell cycle. Thus, in addition to cell density and viabilitycell size is the cellular parameter which should be incorporatednot only into mathematical models of recombinant mammalian cellproduction processes but also into process monitoring andcontrol strategies.     

Stimulation of endothelial cell migration in culture by ladsin,a laminin-5-like cell adhesion protein

Summary Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin α3β1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.     

Diverse effects of the cyclin-dependent kinase inhibitor bohemine: Concentration- and time-dependent suppression or stimulation of hybridoma culture

An analog of aromatic cytokinins, the 2,6,9-trisubstituted purine derivative bohemine, was applied to cultures of mouse hybridoma cells in order to analyze its capacity of suppressing cell growth and maintaining or enhancing the production of monoclonal antibody. Addition of bohemine at concentrations in the range of1–10 μM resulted in a short-term arrest of growth and of monoclonal antibody production. The short-term suppression of cell functions was followed by a significant temporary increase of specific growth rate and of specific production rate. The steady-state viable cell density values, found in semicontinuous cultures, showed a certain stimulation of cell growth in the range of micromolar concentrations of bohemine, and inhibition of growth at 10 and 30 μM concentrations. The profiles of cell cycle phases indicated that hybridoma cells are retarded both at the G₁/S boundary and at the G₂/M boundary, depending on bohemine concentration. The existence of the sequence of events,from suppression to stimulation, suggests that bohemine probably modulates more than one regulatory pathway in the cell. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synergistic effects of focus ultrasound with different frequency and hematoporphyrin on human tumor cells

Human hematopoietic cell K₅₆₂, human melenonla cell LiBr and human stomach cancer cells were exposed to ultrasound (US, 1.75 W/cm², 1.4, 2.16 and 2.4 MHz)in vitro in the presence or absence of hematoporphyrin (Hp, 100 μg/mL). The cell damaging effects of treatments were determined by means of the Trypan Blue dye exclusion test, MTT test and FDA test. The experimental results showed that the same cell line had different sensibilities to the US of different frequencies, and different cell line had different damage at the same acoustical radiation. The cornbined treatment with US and Hp enhanced greatly the cell damage, and no sensibility of insonation cells to US with Hp was observed. The cell damage tests showed that the results of MTT test corresponded well with that of Trypan Blue dye test. Project supported by the Natural Science Foundation of Shaanxi Province     

The modulation of growth of normal rat liver epithelial cells in calcium-poor medium by epidermal growth factor,phenobarbital, phorbol ester,and retinoic acid

Summary The ability of a normal rat liver epithelial cell line with phenotypic characteristics of “oval” cells to grow in calcium-poor medium has been investigated. The growth of these cells could be arrested in medium containing 0.03 mM Ca²⁺, a concentration below which cell necrosis began to occur 24 h postexposure. With increasing calcium concentration, progressive cell proliferation was observed. Epithelial growth factor (EGF) (10 ng/ml) increased the survival and proliferation of cells in calcium-poor medium and the response was inversely correlated with the extracellular calcium concentration. In contrast, phenobarbital (0.2 to 2 mM), 12-0-tetradecanoylphorbol-13-acetate (0.01 to 1 μg/ml), or retinoic acid (0.001 to 0.1 μg/ml) depressed growth of cells in calcium-poor medium. The results confirm the ability of EGF to lower the calcium requirement for proliferation of normal cells, but such an effect does not seem to be a universal property of tumor promoters. This research was supported by National Institutes of Health Grant CA 29323.     

Inactivation of DNA replication origins by the cell cycle regulator, trigonelline, in root meristems of Lactuca sativa

 The effects of trigonelline (TRG) on the cell cycle in root meristems of Lactuca sativa L. were examined in the knowledge that TRG is a cell cycle regulator that causes cell arrest in G2, and prevents ligation of replicons in S-phase. The hypothesis was tested that continuous exposure to TRG would perturb DNA replication which, in turn, would lengthen the cell cycle and impair root elongation. Using DNA fibre autoradiography, mean replicon size was 31 and 13 μm in the TRG (3 mM) and control treatments, respectively. Trigonelline also resulted in a lengthening of both S-phase and the cell cycle and a decrease in primary root elongation. Hence, replicon inactivation was responsible for the protracted S-phase. Trigonelline treatment also resulted in a 1.6-fold increase in fork rate (13.8 μm h⁻¹) compared with the control (8.4 m h⁻¹). The faster fork rate in the larger replicons is in accord with the highly significant positive relationship already established between fork rate and replicon size for various unrelated higher plants. Received: 11 October 1999 / Accepted: 23 December 1999     

Effect of phenylalanine and phenylpropanoids on the accumulation of capsaicinoids and lignin in cell cultures of chili pepper (<Emphasis Type="Italic">capsicum annuum</Emphasis> L.)

Summary Chili pepper (Capsicum annuum L., cv. Tampique?o 74) cell suspensions were employed to study the influence of phenylalanine and phenylpropanoids on the total production of capsaicinoids, the hot taste compounds of chili pepper fruits. The effect of capsaicinoid precursors and intermediates on the accumulation of lignin as an indicator of metabolic diversion was also investigated. Addition of 100 μM of either phenylalanine, cinnamic or caffeic acids to chili pepper cell cultures did not cause significant increases in total capsaicinoids (expressed as capsaicin content, and calculated as averages of the measured values) during the growth cycle. The highest total capsaicinoid content was recorded in cultures grown in the presence of vanillin (142.61 μg g⁻¹ f.wt.), followed by cells treated with 100 μM vanillylamine (104.88 μg g⁻¹ f.wt.), p-coumaric acid (72.36 μg g⁻¹ f.wt.). and ferulic acid (34.67 μg g⁻¹ f.wt.). Capsaicinoid content for control cells was 13.97 μg g⁻¹ f.wt. Chili pepper cell suspensions cultured in the presence of 100 μM of either phenylalanine, or cinnamic, caffeic, or ferulic acids, or the same concentration, of vanillin and vanillylamine, did not exhibit statistically significant differences in the content of lignin as compared with control cells. However, addition of p-coumaric acid (100 μM) to the cultute medium significantly increased thelignin production (c. 10–15 times the contents of control cells).     

Notch3 overexpression causes arrest of cell cycle progression by inducing Cdh1 expression in human breast cancer cells

Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27^Kip accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27^Kip at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27^Kip accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment.     

Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620

N ¹,N ⁴-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N ¹-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 μmol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N ¹-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 μmol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions. This revised version was published online in July 2006 with corrections to the Cover Date.