Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Ultrastructural details of Sertoli cell junctional complexes in vivo and their modifications in tissue culture

Summary This study was carried out using thin sections, lanthanum tracing, and freeze-cleaving in order to investigate the Sertoli cell junctions of rat testis in vivo and their maintenance in culture. The presence of a new type of membrane specialization has been revealed. This consists of a close association between tight junctions and nexuses. The Sertoli cell contact specializations show a progressive disorganization in vitro correlated with the duration of the period in culture. The relationship between the morphological changes in Sertoli cell junctions and the lack of spermatogenesis in culture is discussed.Research performed under the C.N.R. project Biology of Reproduction     

Comparative cytotoxicity of alkyl gallates on mouse tumor cell lines and isolated rat hepatocytes

Alkyl esters of gallic acid inhibited the respiration rate of mouse sarcoma 786A and mouse mammary adenocarcinoma TA3 cell lines and its multiresistant variant TA3-MTX-R more effectively than gallic acid, both in the absence and in the presence of the uncoupler CCCP. The order of inhibition of the respiration rate by gallates in intact cells was n-octyl- approximately iso-amyl- approximately n-amyl- approximately iso-butyl->n-butyl->iso-propyl->n-propyl-gallate>gallic acid. Sarcoma 786A was significantly more susceptible to all seven esters than the TA3 cell line. Respiration rates of the TA3-MTX-R cell line showed almost the same sensitivity to these esters as the TA3 cell line. However, hepatocytes were significantly less sensitive than all tumor cells tested. These alkyl gallates blocked mitochondrial electron flow, mainly at the NADH-CoQ segment, preventing ATP synthesis, which would lead to cellular death. These esters also inhibited, in the same order of potencies as respiration, the growth of 786A, TA3 and TA3-MTX-R cells in culture. In mice carrying TA3 or TA3-MTX-R tumor cells, an important decrease of the tumor growth rate and an increase of survival were observed when mice were treated with iso-butyl gallate alone or in combination with doxorubicin. These results indicate that alkyl gallates are selectively cytotoxic to tumor cells, which may be due to the mitochondrial dysfunctions of these cells.     

Growth and stoichiometry of a Catharanthus roseus cell suspension culture grown under nitrogen-limiting conditions

The uptake of carbohydrate and nitrate by Catharanthus roseus cell suspension cultures was studied in relation to biomass production in shake flasks. Biomass production was similar when using either 6, 12, 18, or 24 mM nitrate as the nitrogen source and 20 g L(-1) sucrose as the carbon source. In all cases, maximum biomass production was reached when carbohydrates were entirely consumer by the cells. Apparent biomass yields, Y(X/S) and Y(X/N) were 0.49 g biomass g(-1) glucose equivalent and 0.23 g biomass mmol(-1) nitrate, respectively. The determination of the cellular carbon-to-nitrogen ration (C/N ration) resulted in the identification of three district growth phases: an active growth phase, and accumulation phase, and a biomass decline phase (endogenous metabolism). The onset of the last two phases was correlated with nitrate and sugar of the last two phases was correlated with nitrate and sugar exhaustion, respectively. Balanced stoichiometric equations describing the active growth and accumulation phases were proposed based on elemental composition and ash content of the biomass. The stoichiometric equation related to the accumulation phase predicts that the available sugars are stored as starch- and lipid-like materials.     

Significances of pH and temperature on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors

Heat-shock protein, glycoprotein 96 (gp96), elicits both innate and adaptive immune responses against tumors or viral infections. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, significances of pH and temperature on the novel bioprocess were studied in stirred-tank bioreactor. Lowering of culture pH and temperature led to a significant reduction of average specific growth rate but cell viability remained high for a prolonged cultivation time resulting in a higher integral of viable cells. Both the maximal viable cell density and gp96 production were attained at a pH of 7.0. Interestingly, gp96 production was increased above and below 37 °C, presumably because gp96 biosynthesis was induced when MethA tumor cell underwent heat or cold. For MethA tumor cell growth 37 °C was desirable, while gp96 production and productivity was obtained at their peak values at 40 °C. The results of this work might be useful to scale-up the bioprocess into the pilot scale.     

Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors

Heat-shock protein glycoprotein (gp96) serves as a natural adjuvant for chaperoning antigenic peptide into the immune surveillance pathway. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, effects of dissolved oxygen tension (DOT) and agitation rate on this process were studied in stirred-tank bioreactor. The optimal conditions for gp96 production were different with those for MethA tumor cell growth. MethA tumor cell growth pattern was not much changed by various levels of DOT and agitation rate, while gp96 biosynthesis was more sensitive to DOT and agitation rate. Compared with 50% of DOT, the production and specific productivity of gp96 was increased by 27 and 66% at 10% of DOT, respectively. Compared with the agitation rate of 100 rpm, the production and volumetric productivity of gp96 was increased by 48 and 144% at the agitation rate of 200 rpm, respectively. Low DOT (i.e., 10% of air saturation) and high agitation rate (i.e., 200 rpm) were identified to be favorable for gp96 biosynthesis. The results of this work might be useful to scale-up the bioprocess into the pilot scale.     

The proliferation of human tumor cell lines in the presence of different agars,agaroses, and methyl cellulose

Summary Human tumor cell lines, derived from cancers of the colon, ovary, and cervix, were grown in liquid tissue culture media and media made semisolid with agar (Bacto & deoxycholate lactose agar), agarose [LE, ME, Sea Plaque and Sea Prep (15/45)], and methyl cellulose. The effects of each agent on overall cell proliferation and rate of overall cell proliferation were examined. The agents, used to make media semisolid, were observed to inhibit or, in some cases, enhance cell growth in a fashion that was characteristic of individual cell lines. These phenomena may be of consequence to the optimization of nutrient media for primary tumor cell preparations. This work was supported by the Veteran's Administration.     

Molecular changes in cell surface membranes resulting from trypsinization of sarcoma 180 tumor cells

Sarcoma-180 tumor cells in culture or grown as an ascites form in the CD-1 mouse have been subjected to mild trypsinization procedures in order to study morphological and molecular changes resulting from proteolysis. The cells attached to a substratum become rounded within 20 min and most undergo cell division, but they do not detach from the substratum. Removal of trypsin permits the cells to go back to their original spindle shape over an 8–20 h period.Surface membranes were isolated from trypsinized ascites and cultured cells and subjected to dodecyl sulfate-acrylamide gel electrophoresis. Both cell types showed the same two kinds of changes in electrophoretic patterns. First, there was a loss of glycoproteins from both cell types, even though they show different complements of cell surface glycoproteins. Second, there is a loss of high molecular weight polypeptides, which have previously been suggested to play a role in membrane stabilization and cell shape. These results further implicate these polypeptides in the control of cell morphology and offer circumstantial evidence for transmembrane interactions of surface glycoproteins with the high molecular weight polypeptides as a factor in controlling cell morphology.     

Covalent binding of 1-nitro-9-(3-dimethyl-n-propylamino) acridine, a new antitumor drug, to DNA of Ehrlich ascites tumor cells in vivo.

After exposing a line of rat liver epithelial cells to a single dose of the carcinogen N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF), a dose-dependent decrease in [³H]uridine incorporation into total cellular RNA was found. Approx. 50% inhibition occurred with 0.5 μg/ml of the compound. The kinetics of the response, the effects of actinomycin D, and the fractionation of the newly synthesized RNA by polyacrylamide gel electrophoresis indicated preferential inhibition of the synthesis of 45S ribosomal RNA precursor and a relative sparing of the synthesis of heterogeneous nuclear RNA.     

Identification and production of flavonoids in a cell suspension culture of Scutellaria baicalensis G.

A high yielding cell line of Scutellaria baicalensis G. has successfully been developed to produce flavonoids. Major components of the flavonoids were identified as baicalin and wogonin-7-O-glucuronic acid by a series of instrumental analyses using UV, IR, MASS, and NMR. After 12 days in suspension culture, the cell growth reached 14 g ^DW/l, and baicalin and wogonin-7-O-glucuronic acid were obtained in concentrations of 2.9 g/l and 1.07 g/l, respectively. The culture temperature was found to be an important parameter for improving production yield of the flavonoids. The yield of baicalin was observed to increase to 4.2 g/l by shifting the temperature from 30 °C to 25 °C after 72 h of suspension culture.Abbreviations DW cell dry weight - FW cell fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - PSH medium phytohormone added Schenk and Hildebrandt medium - FPM a modified Schenk and Hildebrandt medium for flavonoid production     

Quantitation of cell proliferation,colony formation,and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers

Summary A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), γ-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-epoxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.     

HIV‐1 promonocytic and lymphoid cell lines: an in vitro model of in vivo mitochondrial and apoptotic lesion

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV‐1)‐infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro‐mitochondrial or anti‐apoptotic strategies as in vitro cell platforms to deal with HIV‐infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt‐PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial‐nuclear encoded subunits II–IV of cytochrome‐c‐oxidase (COXII‐COXIV), respectively, as well as mitochondrial apoptotic events [voltage‐dependent‐anion‐channel‐1(VDAC‐1)‐content and caspase‐9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1‐assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC‐1 and caspase‐9 content were sharply decreased in both chronic HIV‐1‐infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV‐chronicity, to test novel therapeutic options or to study HIV reservoirs.     

In vitro and in vivo confocal Raman study of human skin hydration: assessment of a new moisturizing agent, pMPC

The hydration capacities of a biomimetic polymer, 2-methacryloyloxethylphosphorylcholine polymer (pMPC), alone and microencapsulated, in association with another well known hydrating polymer, Hyaluronic acid, were investigated in vitro on skin models and in vivo on volunteers by using confocal Raman microspectroscopy. The hydration impact and the relative water content in the Stratum corneum were calculated from the Raman spectra using the OH (water)/CH3 (protein) ratio. Moreover, the follow-up of the presence of pMPC through the Stratum corneum was possible with confocal Raman microspectroscopy, using a characteristic vibration of pMPC, different from that of the encapsulating material. From our in vitro measurements, the improved hydration of the Stratum corneum was confirmed by the use of the encapsulated form of pMPC, which was higher when combined with Hyaluronic acid. On the basis of these in vitro findings, we validated this trend in in vivo measurements on 26 volunteers, and found a good correlation with the in vitro results. Mechanical and ultrastructural studies have been carried out to demonstrate the positive effects of the pMPC on the Stratum corneum function, namely the interaction with lamellar lipids and the plasticizing effects, which are both supposed to spell out the moisturizing effect. This study demonstrates the efficiency of a original hydrating agent, pMPC, entrapped with Hyaluronic acid in a new type of microcapsules by the use of a novel tool developed for both in vitro and in vivo approaches. This indicates a new step to evaluate and improve new moisturizers in response to the cosmetics or dermatologic demands.     

Synthesis,antitumor and antimicrobial activity of some new 6-methyl-3-phenyl-4(3H)-quinazolinone analogues: in silico studies

Some new derivatives of substituted-4(3H)-quinazolinones were synthesized and evaluated for their in vitro antitumor and antimicrobial activities. The results of this study demonstrated that compound 5 yielded selective activities toward NSC Lung Cancer EKVX cell line, Colon Cancer HCT-15 cell line and Breast Cancer MDA-MB-231/ATCC cell line, while NSC Lung Cancer EKVX cell line and CNS Cancer SF-295 cell line were sensitive to compound 8. Additionally, compounds 12 and 13 showed moderate effectiveness toward numerous cell lines belonging to different tumor subpanels. On the other hand, the results of antimicrobial screening revealed that compounds 1, 9 and 14 are the most active against Staphylococcus aureus ATCC 29213 with minimum inhibitory concentration (MIC) of 16, 32 and 32?μg/mL respectively, while compound 14 possessed antimicrobial activities against all tested strains with the lowest MIC compared with other tested compounds. In silico study, ADME-Tox prediction and molecular docking methodology were used to study the antitumor activity and to identify the structural features required for antitumor activity.     

A combination of Buffalo rat liver cell-conditioned medium, forskolin and membrane-bound stem cell factor stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that in vivo

Recent studies have shown that stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the enhancement of cAMP levels increase proliferation and survival of mouse primordial germ cells (PGC) in vitro . Even after the addition of these factors, however, it is still not possible to obtain proliferation of PGC at a rapid rate similar to that in vivo , suggesting the presenge of other growth factor(s) in vivo . We previously reported that tumor necrosis factor-α stimulates proliferation of PGC at earlier migration stages. We now show that the use of SI/SI4-m220 feeder cells and the addition of a medium conditioned with Buffalo rat liver cells and forskolin to the culture medium stimulate PGC obtained from 8.5 days post coitum embryos to proliferate in culture at a rate comparable to that in vivo . Under such conditions, proliferation of PGC continued several days past the timing of growth arrest in vivo ; however, it did stop afterwards. Such proliferating PGC continue to express c-kit and Oct-3 proteins. The characteristics of the culture medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells, suggesting that these rapidly proliferated PGC are not transformed into ES-like EG cells.     

Activity and kinetic properties of basal aryl hydrocarbon hydroxylase during proliferation in the transformable C3H 10T12; CL8 cell line

Basal aryl hydrocarbon hydroxylase (AHH) activity and its kinetic properties were studied as a function of proliferation in C3H mouse embryo 10T$\text{1}\text{2}$ CL8 cells. Activity was low in freshly plated cells, increased during exponential growth, peaked at confluency, and then declined. The apparent K_m-values for benzo[a]pyrene (BP) and NADPH were less in proliferating (approx. 0.37 μM BP, 3.3 nM NADPH) than in confluent cells (0.74–1.39 μM BP, 33.4–53.4 nM NADPH). Cells at different growth states responded differently to benz[a]anthracene (BA) and aminophylline, an inhibitor of cyclic nucleotide phosphodiesterases. When cells were harvested at the mid log phase of growth, 12 h of exposure to aminophylline caused maximum induction, while 24 h of BA treatment were required. In contrast, at early confluence, 12 h of BA treatment gave the greatest levels of activity, while exposure to aminophylline did not induce AHH. In fact, decreases in activity were observed. These differences are indicative of different regulatory mechanisms for BA and aminophylline induction. They also suggest the regulation of basal AHH by cyclic nucleotides changes during growth. The exposure times giving maximum activity were used to determine the kinetic properties of BA-induced activity. As with basal AHH, the K_m-value for BP was less in log phase (0.2–0.4 μM BP) than in confluent cells (0.64–1.05 μM BP). Moreover, the K_m-values for BP and NADPH in control cultures at confluency (0.10–0.14 μM BP, 15.4–23.2 nM NADPH) were less than those for BA-treated cells (0.64 μM BP, 37.9–54.8 nM NADPH) under the same nutritional conditions. The finding that the K_m-value for BP is lower in rapidly dividing cells than in confluent cells may help to explain why proliferating cells are more susceptible to transforming agents.     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.     

Estrogen-induced cyclin D1 and D3 gene expressions during mouse uterine cell proliferation in vivo: Differential induction mechanism of cyclin D1 and D3

D-type cyclins are involved in the regulation of the G₁/S transition of the cell cycle in various cell types cultured in vitro. Little is, however, known about the expression pattern and functional role of D-type cyclins in physiological processes in vivo. In this report, we studied whether the expression of murine D-type cyclins correlates with the states of mouse uterine cell proliferation in vivo. Time-course changes in cyclin D1 and D3 mRNA levels in the uterine tissues of immature mice primed with 17β-estradiol (E₂) were examined by Northern blot hybridization. c-fos and thymidine kinase (TK) mRNA levels were also examined as markers for the transition from G₀ to G₁ and the onset of S phase, respectively. Cyclin D1 and D3 mRNAs were induced 2.5-fold between c-fos and TK mRNA peaks. The E₂-induced cyclin D1 and D3 gene expressions were blocked by antiestrogens tamoxifen and ICI 182,780. We also investigated the effects of cycloheximide (CHX), a protein synthesis inhibitor, on cyclin D1 and D3 gene expressions. When CHX was treated alone, cyclin D3, but not cyclin D1, mRNA was immediately superinduced. The E₂-induced cyclin D3 gene expression was shifted by approximately 6 h when CHX was pretreated 1 hr before E₂ administration. Interestingly, the ³H-thymidine incorporation experiment showed that the mouse uterine cell cycle progression also shifted by 6 hr with pretreatment of CHX. The overall results suggest that both cyclin D1 and D3 mRNAs are constitutively expressed in uterine tissues and induced by E₂ at G₁ phase of the mouse uterine cell cycle. However, the superinducibility and temporal shift of cyclin D3 by CHX suggest that there is a different regulatory mechanism underlying cyclin D1 and D3 gene expressions in the mouse uterine cell cycle progression. Mol. Reprod. Dev. 46:450–458, 1997. © 1997 Wiley-Liss, Inc.