Cell Viability Growth Research Articles

Methylenetetrahydrofolate Reductase C677T Polymorphism, Folic Acid and Riboflavin Are Important Determinants of Genome Stability in Cultured Human Lymphocytes

We tested the hypothesis that methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, folic acid deficiency and riboflavin deficiency, independently or interactively, are important determinants of genomic stability, cell death, cell proliferation and homocysteine (Hcy) concentration in 9-d human lymphocyte cultures. Lymphocytes of seven wild-type (CC) and seven mutant (TT) homozygotes were cultured under the four possible combinations of deficiency and sufficiency of riboflavin (0 and 500 nmol/L) and folic acid (20 and 100 nmol/L) at a constant L-methionine concentration of 50 micromol/L. Viable cell growth was 25% greater in TT than in CC cells (P<0.05) and 32% greater at 100 nmol/L folic acid than at 20 nmol/L folic acid (P=0.002). The comprehensive cytokinesis-block micronucleus assay was used to measure micronuclei (MNi; a marker for chromosome breakage and loss), nucleoplasmic bridges (NPB; a marker of chromosome rearrangement) and nuclear buds (NBUD, a marker of gene amplification). The MNi levels were 21% higher in TT cells than in CC cells (P<0.05) and 42% lower in the high folic acid medium than in the low folic acid medium (P<0.0001). The NBUD levels were 27% lower in TT cells than in CC cells (P<0.05) and 45% lower in the high folic acid medium than in the low folic acid medium (P<0.0001). High riboflavin concentration (500 nmol/L) increased NBUD levels by 25% (compared with 0 nmol/L riboflavin) in folate-deficient conditions (20 nmol/L folic acid medium; P<0.05), and there was an interaction between folic acid and riboflavin that affected NBUD levels (P=0.042). This preliminary investigation suggests that MTHFR C677T polymorphism and riboflavin affect genome instability; however, the effect is relatively small compared with that of folic acid.

Cancer cells surviving hypoxia obtain hypoxia resistance and maintain anti-apoptotic potential under reoxygenation.

In solid tumors hypoxia and reoxygenation may be important factors in secondary expansion after anti-cancer therapy. Our study examined the effect of hypoxia and reoxygenation on the apoptotic potential of cancer cells. Four experimental groups were studied using a human colorectal cancer cell line (HCT116) that is apoptosis-competent in conventional culture: (1) sham, cells grown under conventional conditions; (2) hypoxic, cells cultured in 95% N2 and 5% CO2 for 24 hr; (3) continued hypoxic, cells cultured for 48 hr; and (4) reoxygenation, cells grown in hypoxic conditions for 24 hr followed by another 24 hr under conventional conditions. Protein expression of p53, bcl-2 and PCNA were determined by immunohistochemistry and immunoblotting (p53), and viable cell growth rate was determined. Hypoxia for 24 hr induced significant up-regulation of p53 and bcl-2 expression, accompanied by significant decreases of cell growth rate and PCNA expression. Up-regulation of p53 and bcl-2 expression persisted with both continued hypoxia and reoxygenation, despite increased cell growth rate and PCNA expression. Cells escaping hypoxia acquired sustained resistance to apoptosis and proliferate despite an elevated p53 level, suggesting that p53 transfer to hypoxic solid tumor should be reevaluated as a cancer gene therapy approach.

The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Culture redox potential (CRP) and oxygen uptake rate (OUR) were monitored on-line during glucose- and glutamine-limited batch cultures of a murine hybridoma cell line that secretes a neutralizing monoclonal antibody specific to toxin 2 of the scorpion Centruroides noxius Hoffmann. It was found that OUR and CRP can be used for assessing the viable cell concentration and growth phases of the culture. Before nutrient depletion, OUR increased exponentially with viable cell concentration, whereas CRP decreased monotonically until cell viability started to decrease. During the death phase, CRP gradually increased. A sudden decrease in OUR occurred upon glucose or glutamine depletion. CRP traced the dissolved oxygen profile during a control action or an operational eventuality, however, during nutrient depletion it did not follow the expected behavior of a system composed mainly by the O2/H2O redox couple. Such a behavior was not due to the accumulated lactate or ammonia, nor to possible intracellular redox potential changes caused by nutrient depletion, as inferred from respiration inhibition by rotenone or uncoupled respiration by 2,4-dinitrophenol. As shown in this study, operational eventualities can be erroneously interpreted as changes in OUR when using algorithms based solely on oxygen balances. However, simultaneous measurements of CRP and OUR may be used to discriminate real metabolic events from operational failures. The results presented here can be used in advanced real-time algorithms for controling glucose and glutamine at low concentrations, avoiding under- or over-feeding them in hybridoma cultures, and consequently reducing the accumulation of metabolic wastes and improving monoclonal antibody production. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 555–563, 1997.

The use of culture redox potential and oxygen uptake rate for assessing glucose and glutamine depletion in hybridoma cultures

Culture redox potential (CRP) and oxygen uptake rate (OUR) were monitored on-line during glucose- and glutamine-limited batch cultures of a murine hybridoma cell line that secretes a neutralizing monoclonal antibody specific to toxin 2 of the scorpion Centruroides noxius Hoffmann. It was found that OUR and CRP can be used for assessing the viable cell concentration and growth phases of the culture. Before nutrient depletion, OUR increased exponentially with viable cell concentration, whereas CRP decreased monotonically until cell viability started to decrease. During the death phase, CRP gradually increased. A sudden decrease in OUR occurred upon glucose or glutamine depletion. CRP traced the dissolved oxygen profile during a control action or an operational eventuality, however, during nutrient depletion it did not follow the expected behavior of a system composed mainly by the O(2)/H(2)O redox couple. Such a behavior was not due to the accumulated lactate or ammonia, nor to possible intracellular redox potential changes caused by nutrient depletion, as inferred from respiration inhibition by rotenone or uncoupled respiration by 2,4-dinitrophenol. As shown in this study, operational eventualities can be erroneously interpreted as changes in OUR when using algorithms based solely on oxygen balances. However, simultaneous measurements of CRP and OUR may be used to discriminate real metabolic events from operational failures. The results presented here can be used in advanced real-time algorithms for controling glucose and glutamine at low concentrations, avoiding under- or over-feeding them in hybridoma cultures, and consequently reducing the accumulation of metabolic wastes and improving monoclonal antibody production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 555-563, 1997.

Are laryngeal papilloma virus-infected cells viable in the plume derived from a continuous mode carbon dioxide laser, and are they infectious? A preliminary report on one laser mode

The purpose of this study was to determine the potential risk of transmitting viable viral infected cells as well as viral infectivity of laryngeal papilloma in the plume derived from a continuous mode carbon dioxide laser. Each of 10 juvenile recurrent laryngeal papilloma specimens was divided into two equal parts, and one part was irradiated with a carbon dioxide laser employing a continuous mode at the power setting of 10 watts with 0.5 mm spot size and a power density of 1667 W/cm2. The resultant laser plume was trapped and was cultured simultaneously with the other part of the specimen which served as the control. All irradiated specimens tested yielded negative culture results while all the control counterparts revealed viable cell growth. To detect the viral infectivity, laser plume was cultured with two separate cell systems, one was the porcine PS clone D cell line and the other normal mucosal cells obtained from the same patient, and to control these systems both cell lines were also designed to be infected with polio virus. Both cell lines in the viral infectivity testing systems revealed no sign of viral infection. The results suggest that papilloma virus-infected cells cannot survive the continuous mode of carbon dioxide laser irradiation. We primarily conclude that, to avoid airborne transmission of plume containing laryngeal papilloma viral-infected cells and infectious viral particles, the carbon dioxide laser parameters should be in a continuous mode with the power density equal to, or more than, 1667 W/cm2.

Secretion of recombinant proteins from hydroxyethyl methacrylate-methyl methacrylate capsules

Current human gene therapy relies on genetic modification of the patient's own cells. An alternate nonautologous approach is to use universal cell lines engineered to secrete therapeutic products. Protection with immunoisolation devices before implantation would allow the use of the same recombinant cell line for treating different patients, thus potentially lowering the cost of treatment. To study the properties of a mechanically stable synthetic biomaterial, hydroxyethyl methyacrylate-methyl methacrylate (HEMA-MMA) as the immuno-isolation device, we encapsulated recombinant mouse fibroblast cells engineered to secrete products ranging from 27 to 300 kDa in size (human growth hormone, mouse beta-hexosaminidase and beta-glucuronidase) in the presence or absence of the extracellular matrix Matrigel. Both viability and cell number in the microcapsules increased with time after encapsulation and cell morphology indicated viable cell growth, thus showing that the capsule membrane barrier was compatible with nutrient/waste exchange necessary for normal metabolic activity.The intracellular levels of these recombinant gene products were constant throughout the experimental period of 22 days in the presence or absence of Matrigel, thus demonstrating that the microenvironment did not lead to downregulation of the transgenes. However, the extracellular levels of the gene products secreted from the cells and trapped within the microcapsules were dependent on the molecular size of the product and presence of Matrigel. With the 27-kDa human growth hormone, the presence of Matrigel caused its retention within this intracapsular space, but its release from the microcapsules to the culture medium was not impeded. With the 120-kDa beta-hexosaminidase or the 300-kDa beta-glucuronidase, they were retained within the microcapsule space regardless of the presence or absence of Matrigel, and their passage from the microcapsules to the media was totally blocked. In conclusion, the HEMA-MMA microcapsules are supportive of recombinant cell growth and maintained their molecular cutoff at approximately 100 kDa. Inclusion of extracellular matrix was unable to improve cell growth and may impede the exit of some gene products.

Secretion of recombinant proteins from hydroxyethyl methacrylate‐methyl methacrylate capsules

Current human gene therapy relies on genetic modification of the patient's own cells. An alternate nonautologous approach is to use universal cell lines engineered to secrete therapeutic products. Protection with immunoisolation devices before implantation would allow the use of the same recombinant cell line for treating different patients, thus potentially lowering the cost of treatment. To study the properties of a mechanically stable synthetic biomaterial, hydroxyethyl methyacrylate-methyl methacrylate (HEMA-MMA) as the immuno-isolation device, we encapsulated recombinant mouse fibroblast cells engineered to secrete products ranging from 27 to 300 kDa in size (human growth hormone, mouse β-hexosaminidase and β-glucuronidase) in the presence or absence of the extracellular matrix Matrigel. Both viability and cell number in the microcapsules increased with time after encapsulation and cell morphology indicated viable cell growth, thus showing that the capsule membrane barrier was compatible with nutrient/waste exchange necessary for normal metabolic activity. The intracellular levels of these recombinant gene products were constant throughout the experimental period of 22 days in the presence or absence of Matrigel, thus demonstrating that the microenvironment did not lead to downregulation of the transgenes. However, the extracellular levels of the gene products secreted from the cells and trapped within the microcapsules were dependent on the molecular size of the product and presence of Matrigel. With the 27-kDa human growth hormone, the presence of Matrigel caused its retention within this intracapsular space, but its release from the microcapsules to the culture medium was not impeded. With the 120-kDa β-hexosaminidase or the 300-kDa β-glucuronidase, they were retained within the microcapsule space regardless of the presence or absence of Matrigel, and their passage from the microcapsules to the media was totally blocked. In conclusion, the HEMA-MMA microcapsules are supportive of recombinant cell growth and maintained their molecular cutoff at ∼100 kDa. Inclusion of extracellular matrix was unable to improve cell growth and may impede the exit of some gene products. © 1996 John Wiley & Sons, Inc.

Evidence that cell survival is controlled by interleukin‐3 independently of cell proliferation

Hemopoietic cell proliferation is controlled by a set of polypeptide growth factors and regulatory molecules that bind to cell surface receptors inducing cellular responses. Maintenance of a viable state, cell growth, DNA synthesis and mitosis are basic properties of proliferating cells, but links between growth factor receptors and each of these cellular outcomes are poorly understood. Most studies have monitored DNA synthesis as a measure of progression through the cell cycle or directly measured viable cell numbers, but cell survival per se as an output of receptor activation by ligand, has received little attention. In this study we have used a bone marrow-derived murine cell line that is dependent on interleukin-3 for growth, to investigate the relationship between DNA synthesis and a biochemical marker of cell survival, reduction of the tetrazolium salt, MTT. We show that at times up to 6 hr, continued DNA synthesis, RNA synthesis, protein synthesis, and mitochondrial respiration are not necessary for background or IL-3-stimulated MTT reduction. Furthermore, dibutyryl cyclic AMP promoted background and IL-3-dependent MTT reduction while simultaneously inhibiting DNA synthesis. These results provide evidence that IL-3 controls events involved in MTT reduction and cell survival independently of DNA synthesis.

Enhancement of monoclonal antibody yield by hybridoma fed‐batch culture, resulting in extended maintenance of viable cell population

Hybridoma batch cultures were extended using feed formulations based on nutrient consumption measured during different batch culture phases when (a) growth but negligible antibody production was taking place; (b) maximum antibody production rate and declining viable cell growth rate were observed. Strategy (a) was the more successful (2.8-fold compared with 1.8-fold antibody titer increase) and maintained cell viability for longer. Analysis of the effects of omitting individual amino acids yielded results which were consistent with those from the feeding experiment.

An investigation of the diffusion‐limited growth of animal cells around single hollow fibers

To compare modeling with experimental data of cell growth surrounding individual fibers, the growth profiles of hybridoma cells in the extracapillary space of single hollow fiber bioreactors were examined. Agarose was provided in the extracapillary space to provide support and minimize convection. By sacrificing bioreactors at various time intervals, the growth profiles of cells surrounding a single hollow fiber could be monitored with increasing time. Using photomicroscopy and viable staining, areas of viable and nonviable cell growth were examined at various stages of development ranging from initial seeding to stable growth conditions. Cells were found to act as nucleation sites for the growth of individual colonies within the agarose. Profiles at stable growth conditions resulted in a thick cell mass near the surface of the fiber wall followed by cell colonies of decreasing size with increasing radial distance. A simplified theoretical model for cell growth was developed using mass balance equations for substrate penetration into individual cell colonies as well as away from the wall of a single fiber. The resulting profiles derived from theory were compared with experiments and found to be in good agreement for entering oxygen concentrations of 5% and 20%.

Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines.

The t(14;18) of human follicular B cell lymphoma translocates the Bcl-2 gene into the Ig H chain locus and markedly deregulates Bcl-2 expression. We sought to determine if Bcl-2 could be directly implicated in a growth-factor pathway. Consequently, we introduced a retrovirus containing the murine Bcl-2 gene (N2-M-Bcl-2) or the parental retrovirus (N2) into a series of factor-dependent hemopoietic cell lines. Overexpressed Bcl-2 resulted in no long term IL-2, IL-3, or IL-6 independent clones, indicating that Bcl-2 could not spare the need for a specific ligand-receptor interaction. However, Bcl-2 did extend the short term survival of IL-3-dependent cell lines after factor deprivation. Although viable, IL-3-deprived pro B lymphocytes (FL5.12) bearing N2-M-Bcl-2 were in Go, and deregulated Bcl-2 did not obviously influence cell-cycle progression. Bcl-2 predominant effects were to delay the onset of cell death and to modestly augment viable cell growth in the first 48 h after IL-3 deprivation. This death sparing was associated with increased levels of Bcl-2 RNA and protein in factor-deprived cells possessing N2-M-Bcl-2. This result was not restricted to prolymphocytes because an IL-3-dependent mast cell line (32D) as well as a promyeloid line (FDC-P1) demonstrated the same response to Bcl-2. Moreover, the effect was not limited to the IL-3/IL-3R signal transduction pathway in that promyeloid cells maintained in granulocyte-macrophage-CSF or IL-4 displayed a similar response. Yet, Bcl-2-enhanced cell survival was not universal as an IL-2-dependent T cell line, and an IL-6-dependent myeloma line demonstrated no consistent effect upon IL withdrawal. Thus, Bcl-2 appears to interfere with cell death but in a cell type and/or factor-restricted fashion.

APhA Policy Committee Reports

Thin films of zinc oxide (ZnO) were produced by reactive DC magnetron sputtering with incidence angles of α = 0°, 40°, 60° and 80° (glancing angle deposition configuration), aiming to be tested against the opportunist pathogenic fungus Candida albicans. The results showed the formation of stoichiometric ZnO thin films, with inclined columns for incidence angles equal to or >40°. All thin films presented high transparency in the visible range above the bandgap region (near 380 nm). The deposition conditions gave rise to a three-fold increase of the surface porosity with the increment of the incidence angle. Noteworthy, is the formation of different types of pores distributions, from micro-, through meso-, to macropores. Regarding the biological effect, the thin films produced with inclined columns presented a significant antifungal activity, with the inhibition of viable cell growth by 68%. Moreover, the formation of mesoporous films enhanced the antifungal properties of ZnO thin films against Candida albicans. The overall behaviour indicates that these thin films are promising candidates to be applied in antimicrobial surfaces, as well as to be used in further studies to determine the molecular mechanisms involved in the antimicrobial action of ZnO.

Effect of HTLV-III on the macromolecular synthesis in HTLV-I carrying cell line, MT-4.

Upon infection of human T-cell leukemia virus type I (HTLV-I)-carrying human T-cell lines such as MT-4, HTLV-III, a probable etiologic agent of acquired immune deficiency syndrome (AIDS) caused fast and strong cytopathic effects leading ultimately to the death of the cells. Such effects were preceded by the rapid induction of HTLV-III antigens. Cell lines not infected with HTLV-I could, however, be subcultured after infection with HTLV-III, although they were also positive for HTLV-III antigens. To understand this cytopathogenicity of HTLV-III in HTLV-I bearing cells, macromolecular synthesis, including DNA synthesis and total protein synthesis, and also IL-2 receptor expression were investigated kinetically. In infected MT-4 cells DNA synthesis was markedly inhibited by HTLV-III after the HTLV-III antigen synthesis became evident. This inhibition occurred before cell damage was detected in terms of viable cell-growth, but after induction of HTLV-III antigen. Puromycin, at 40 micrograms/ml, caused no toxic changes in MT-4 cells over 3 days but prevented viral antigen synthesis and virus-induced cytopathic effect. Protein synthesis and IL-2 receptor expression were also inhibited at 4 and 5 days post infection. The degree of the effects and their kinetics suggest that they are the secondary effects of cytotoxicity by HTLV-III infection.

Differential Chemotherapeutic Susceptibility of Human T-Lymphocytes and B-Lymphocytes in Culture2

After previous work from this laboratory revealed that asparaginase was 800-2,000 times more inhibitory against human T-lymphocytes in culture than against B-lymphocytes, a similar further study of 13 chemotherapeutic and immunosuppressive agents was done. Cytosine arabinoside and 5-fluorouracil also had differential inhibitory activities on human T- and B-cells in culture. On the basis of the dose producing 50% inhibition of viable cell growth on day 5, cytosine arabinoside had 45-80 times more inhibitory activity against T-cells than against B-cells. In contrast to asparaginase and cytosine arabinoside, 5-fluorouracil had 10-20 times more inhibitory activity against B-cells. The rest of the chemotherapeutic and immunosupressive agents tested had minor or no differential activity. These findings indicated that T-cell response to asparaginase and cytosine arabinoside and B-cell response to 5-fluorouracil may be exploitable for the differential immunosuppressive effects presumed to be active in vivo. In addition, such differential responses may predict differential tumor cell behavior against these chemotherapeutic agents by T- and B-cell neoplasms in vivo.

Suppression of amphotericin B methyl ester induced growth stimulation in intraspecific mouse somatic cell hybrids.

Amphotericin B methyl ester (AME), the chemically modified derivative of amphotericin B, induced a concentration-dependent growth stimulatory effect on B82 mouse cells as indicated by increased 24- and 72-hour viable cell number, growth rate and DNA and RNA synthesis. In contrast, AME was not growth promoting toward RAG mouse cells or B82-RAG somatic cell hybrids, while hybrid cells exhibited the increased AME resistance pattern of B82 parental cells. A dissociation between the phenotypic expression of growth stimulation and polyene sensitivity was demonstrated in intraspecific mouse hybrids.

Effect of 5-bromodeoxyuridine on hyaluronic acid synthesis of a clonal hybrid line of mouse and chinese hamster in culture.

AbstractA clonal hybrid line, B‐6, derived from the fusion of mouse and Chinese hamster cell lines, has an inheritable ability to synthesize and secrete hyaluronic acid (HA). In order to elucidate the control mechanism of expression of this differentiated function, we studied the effect of 5‐bromodeoxyuridine (BUDR), a thymidine (TDR) analog on the growth and HA synthesis of this hybrid line. The presence of BUDR at 0.5–1 μg/ml of medium had no effect on cell multiplication, but reduced HA production by approximately 25 to 40%. Treatment with 5 to 25 μg/ml of the analog resulted in about 15% decrease of the total cell growth, or 30% decrease of the viable cell growth, while mucopoly‐saccharide production was reduced by more than 70%. These results indicate that BUDR affects HA synthesis more strongly than it does cell growth.Inhibition was found to be detectable after two days of culture with BUDR. This effect was prevented by the simultaneous addition of TDR in a concentration more than twice that of the analog. Furthermore the inhibitory effect was reversible, since a gradual but complete recovery of HA synthesis occurred after removal of BUDR from the medium. In contrast, the growth rate of B‐6 cells was discovered to be more sensitive than HA synthesis to the inhibitory action of either excess TDR or another analog, 5‐fluorodeoxyuridine (FUDR).