Properties of the H-4-II-E tumor cell system. II. In vitro characteristics of an experimental tumor cell line.

The growth and cell proliferation characteristics of the H-4-II-E cell line, giving rise to hepatoma H-4-II-E when inoculated into male ACI rats, were studied in vitro. Following seedling of 2 x 10(5) cells into culture dishes, exponential cell growth occurs in cultures fed both at 24 hr and 48 hr intervals with a population doubling time of 18-4 hr. Plateau phase growth conditions are established on day 7 and day 5 for cultures fed at 24 hr and 48 hr intervals respectively. Both the plateau phase cell density and the maintenance of plateau phase appear dependent on the frequency of feeding. For cultures fed daily, the transition from exponetial growth to plateau phase results from both a reduction in the number of proliferating cells (99% v. 35%) as well as an elongation of the cell cycle (17-7 hr v. 128-4 hr). The cell proliferation characteristics of the culture are further discussed in reference to both cell growth and feeding schedules of other cell lines.     

Isoniazid Uptake in Relation to Growth Inhibition of Mycobacterium tuberculosis

(14)C-isoniazid (INH) was used to study the relationship between drug uptake or binding by Mycobacterium tuberculosis and growth inhibition of the organism, which is dependent upon the concentration of drug and the duration of exposure. When strain H37R(a), grown in modified Sauton's liquid medium, was treated with 0.1 mug of INH per ml for 2 to 6 hr, followed by 10 mug of nicotinic hydrazide (NH) per ml to block further INH uptake, growth was retarded but not completely inhibited upon continued incubation. NH itself did not retard growth. However, cells treated in a similar manner with INH alone grew normally when diluted 1:100 in fresh, drug-free media. Uptake data showed that bacilli exposed to 0.1 mug of INH per ml accumulated 5.5, 9.7, and 12 mmug/mg of dry cells at 2, 4, and 6 hr, respectively. Other experiments suggested that once isoniazid is bound, it is not rapidly lost when NH is added or when the cells are diluted in fresh media. In the presence of 1.0 mug of INH per ml, tubercle bacilli took up 10 to 37 mmug/mg of dry cells in 20 to 90 min. These cells were not markedly inhibited when diluted 1:40 in fresh NH-containing media and incubated for 6 days. Growth inhibition of tubercle bacilli by INH depends on the uptake of sufficient drug, but the evidence obtained in this study suggests that the absolute concentration of bound INH is not as important in the action of the drug as is the maintenance of a critical cellular concentration for a requisite period of time.     

Theileria parva: cell culture analysis of clones of macroschizont-infected bovine lymphoblastoid cells

Three clones (H7, D7, and C5) were established from single cells of a bovine lymphoblastoid cell line (IR.TPM.1) infected with macroschizonts of the protozoan parasite Theileria parva. The cloning efficiency using feeder layers was 0.3–0.4. The mean parasite size (the number of parasite nuclei per cell) was different in each clone and was correlated to the growth rate. The fast growing clone, C5 (population doubling time 24 hr), contained smaller (mean parasite nuclear number, 12) parasites than a slow growing clone, D7 (population doubling time, 73 hr; mean number of parasite nuclei per cell, 35.3). The third clone, H7, had an intermediate growth rate (population doubling time, 49 hr) and parasite size (mean nuclei number, 18.1). There was variation in the incidence of microschizonts among the clones but microschizont-free clones were not isolated. When the clones were subjected to 4.3 × 10^?7M aminopterin, 20–25% of the cell population of clones H7 and C5 and the uncloned parent line lost their parasites in 4 days, while it took 7 days to reach a similar result (31% parasite-free cells) in clone D7. We were unable to isolate parasite-free clones from cells treated with aminopterin. Hydroxyurea (4 × 10^?4M) inhibited the growth of clone C5, but the macroschizonts continued to proliferate, and the incidence of cells with microschizonts increased. The size profile analysis showed that most of the aminopterin-treated cells were 9.0 μm, the hydroxyurea-treated cells 14.7 μm, and the untreated cells 10.8 μm in diameter.     

Inhibition of HeLa-S3 cell proliferation and biosynthesis by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)

Treatment of HeLa-S3 cells in suspension cultures with 60 microM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) for 18-30 hr stops the growth of the cell population when treatment is carried out at 37 degrees C in Eagle's spinner culture medium supplemented with 5% fetal bovine serum. The length of the period of no growth after termination of treatment is directly related to the duration of DRB treatment. Upon resumption of growth, the rate becomes exponential and is not distinguishably different from the control rate (doubling time: 19 hr). The growth of the progeny population of the previously DRB-treated cells is as sensitive to inhibition by DRB as the growth of control populations not treated with DRB. After treatment of cells with DRB for 30 hr at 39.5-40 degrees C, the population which grows out has a prolonged doubling time. DRB treatment at 37 degrees C for 5 hr markedly inhibits uridine uptake and cellular RNA synthesis in the presence either of 5 or 15% serum. After treatment for 48 hr in 15% serum, inhibition of RNA synthesis by DRB is significantly decreased. DRB treatment does not inhibit leucine uptake in HeLa cells growing in suspension cultures. Protein synthesis is moderately inhibited in 5% serum and only slightly inhibited in 15% serum after either 5- or 48-hr period of treatment.     

Cell cycle changes during neural crest cell differentiation in vitro.

Chick trunk neural tubes containing neural crest cells were cultured in vitro. Cell outgrowth from these neural tube explants consists primarily of a small stellate cell population. After 3 days in culture the small stellate cell population undergoes a remarkable change in morphology that is characterized by a more refractile appearance in the phase contrast microscope. Subsequent to this change in morphology, pigment granules become visible in the cytoplasm after 4 days in culture. After 6 days in culture, virtually all of the small stellate cells are pigmented. The cell cycle parameters of the small stellate cell population are: S = 4.4 ± 1.2 hr (SD). G2 = 1.5 ± 1.0 hr (SD). M = 1.7 ± 0.6 hr (SD). and Gl = 3.8 ± 1.0 hr (SD). Continuous label experiments demonstrate that (G1+G2+M) increases from 7 hr in Day 4 cells, as yet unpigmented, to 12 hr in Day 5 cells that have become pigmented. This change is consistent with an increase in G1 and/or G2 that is closely correlated with the appearance of pigment granules. It is of interest that this cell cycle change is correlated with a rather late event in the developmental program of these neural crest cells rather than with the earlier morphological change observed after 3 days in culture.     

Lymphocyte ecto-5'-nucleotidase activity in infancy: increasing activity in peripheral blood B cells precedes their ability to synthesize IgG in vitro

Ecto-5'-nucleotidase activity was measured in peripheral blood lymphocytes isolated from serial specimens from nine healthy full-term infants and two premature infants at 0, 2, 4, and 6 mo of age. The postnatal nadir in activity was 7.1 +/- 2.0 nmol/hr/10(6) cells, which is the same as the activity in cord blood lymphocytes (7.0 +/- 2 nmol/hr/10(6) cells). The activity rose twofold to 13.2 +/- 3.8 nmol/hr/10(6) cells at 6 mo of age (p less than 0.001, paired t-test), which is similar to the activity in adult peripheral blood lymphocytes (14.1 +/- 6.3 nmol/hr/10(6) cells). This increased activity in total lymphocytes reflects increased activity in the B cell population. B cell ecto-5'-nucleotidase activity in two infants at 12 to 13 mo of age was 19.3 and 25.2 nmol/hr/10(6) cells, values that are four-to fivefold higher than for cord blood B cells (5.6 +/- 2.8 nmol/hr/10(6) cells) and within the normal range for adult B cells (27.9 +/- 12 nmol/hr/10(6) cells). In spite of a greatly expanded peripheral blood B cell population, studies of immunoglobulin biosynthesis in vitro demonstrated that infant peripheral blood B cells are functionally immature with no synthesis of IgG in response to Epstein Barr virus. Thus, the increase in peripheral blood B lymphocyte ecto-5'-nucleotidase activity in infants precedes their acquisition of a capacity for IgG synthesis in vitro. Data from a hypogammaglobulinemic infant revealed a persistently low ecto-5'-nucleotidase activity over a 10-mo period until at 14 mo of age the activity was 8.8 nmol/hr/10(6) cells in total lymphocytes and 13.0 nmol/hr/10(6) cells in B cells, which correlated with in vivo and in vitro evidence of delayed B cell maturation. Thus, ecto-5'-nucleotidase activity may be a useful cell surface marker in studies of human postnatal B cell maturation.     

CHANGES IN CELL PROLIFERATION KINETICS OCCURRING DURING THE LIFE HISTORY OF MONOLAYER CULTURES OF A MOUSE TUMOUR CELL LINE

Following inoculation of monolayer cultures of EMT6 mouse tumour cells at 10⁵ cells, a short lag is followed by 3 days of exponential growth with a population doubling time of 12 hr. A plateau cell number is reached between days 4 and 5 and is maintained for at least 8 days. During exponential growth, the pulse ³H-TdR labelling index is 55–60%, all cells are in cycle, and the median cycle time is 11–12 hr. For the first 3 days of plateau phase, the labelling index is about 25 % and there is considerable cell loss. The cell cycle is 32–40 hr, and S-phase is very long. Later in plateau phase, the labelling index falls to <2 % and there is little cell loss. The changes in kinetics occurring in EMT6 cultures are discussed with reference to reported changes occurring in other cell lines.     

Changes in purine nucleoside phosphorylase activity during thymosin-induced human null cell differentiation

Purine nucleoside phosphorylase (PNP) is a purine salvage pathway enzyme which we have found to be 8-10 times more active (per cell) in human peripheral blood null lymphocytes than in T lymphocytes. To test the hypothesis that null cells are, in part, pre-T lymphocytes we have defined an in vitro system for null cell differentiation into T cells and examined PNP activity during this differentiation process. We found that about 10% of human null cells could be driven to differentiate into T cells using thymosin fraction 5 (TF5) an extract of bovine thymus glands. The response to TF5 was dose related to up to 250 micrograms/ml with a maximum response occurring by 42-46 hr incubation. Exposure to TF5 was necessary for more than 4 hr but no more than 8 hr in order to obtain a maximum response. Both OKT4 and OKT8 positive cells were present in the newly differentiated T cell population but OKT8 positive cells appeared to predominate (OKT4/OKT8 = 0.698 +/- 0.30, mean +/- 1 SD). The differentiation process did not involve DNA synthesis but was inhibited at 4 degrees C. In the newly differentiated T cells PNP activity per cell was 8- to 10-fold lower (36 +/- 23 nm/hr/106 cells) than in null cells (311 +/- 136), and was at a level similar to mature T cells (56 +/- 7). Thus, human peripheral blood null cells can be induced to differentiate into T lymphocytes which can be characterized by both surface markers and biochemical parameters. Future studies will look at the function of TF5-induced T cells and the regulation of PNP activity during the differentiation process.     

Synchronization of Drosophila cells in culture.

We synchronized Drosophila cell lines (Schneider's line 2 and Kc) by allowing the cells to enter the stationary phase of growth and then diluting them into fresh culture medium. The cells of both cell lines entered S phase, after an 8- to 14-hr delay, in a state of partial synchrony; 60 to 80% of the cell population accumulated in S phase. Measurements of the cell cycle phases of Schneider's line 2 cells (S = 14 to 16 hr; G2 = 6 to 8 hr; M = 0.4 hr) were similar to those of Kc cells.     

Dynamics of circulating concentrations of gonadotropins and ovarian hormones throughout the menstrual cycle in the bonnet monkey: role of inhibin A in the regulation of follicle‐stimulating hormone secretion

In higher primates, increased circulating follicle‐stimulating hormone (FSH) levels seen during late menstrual cycle and during menstruation has been suggested to be necessary for initiation of follicular growth, recruitment of follicles and eventually culminating in ovulation of a single follicle. With a view to establish the dynamics of circulating FSH secretion with that of inhibin A (INH A) and progesterone (P₄) secretions during the menstrual cycle, blood was collected daily from bonnet monkeys beginning day 1 of the menstrual cycle up to 35 days. Serum INH A levels were low during early follicular phase, increased significantly coinciding with the mid cycle luteinizing hormone (LH) surge to reach maximal levels during the mid luteal phase before declining at the late luteal phase, essentially paralleling the pattern of P₄ secretion seen throughout the luteal phase. Circulating FSH levels were low during early and mid luteal phases, but progressively increased during the late luteal phase and remained high for few days after the onset of menses. In another experiment, lutectomy performed during the mid luteal phase resulted in significant decrease in INH A concentration within 2 hr (58.3±2 vs. 27.3±3 pg/mL), and a 2‐ to 3‐fold rise in circulating FSH levels by 24 hr (0.20±0.02 vs. 0.53±0.14 ng/mL) that remained high until 48 hr postlutectomy. Systemic administration of Cetrorelix (150 µg/kg body weight), a gonadotropin releasing hormone receptor antagonist, at mid luteal phase in monkeys led to suppression of serum INH A and P₄ concentrations 24 hr post treatment, but circulating FSH levels did not change. Administration of exogenous LH, but not FSH, significantly increased INH A concentration. The results taken together suggest a tight coupling between LH and INH A secretion and that INH A is largely responsible for maintenance of low FSH concentration seen during the luteal phase. Am. J. Primatol. 71:817–824, 2009. © 2009 Wiley‐Liss, Inc.     

Analysis of population cytokinetics of chick myocardial cells in tissue culture

The growth of embryonic chick cardiac myocytes and fibroblasts in tissue culture was evaluated by the kinetics of nuclear labeling during continuous exposure to [3H]thymidine. The fraction of mitotically active cells, the mean intermitotic period and the population doubling times were determined in each cell type during 3 weeks in culture. After 24 hr in culture, 90% of the muscle cells were mitotically active with minimal population doubling times of 65 hr. By 17 days in culture only 5% of the myocytes continued to divide with population doubling times greater than 3000 hr. Primarily, the lengthening of doubling times was due to a withdrawal of cells from the mitotic cycle and much less to a lengthening of the intermitotic period. Growth of cardiac muscle cells from embryonic hearts from 4 to 10 days of development was also compared. Muscle cells from younger hearts displayed greater mitotic activity than those from older hearts at equivalent times in culture.     

Analysis of the growth kinetics of a human lymphoma cell line

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 X 10(4)--5 X 10(5) cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with 3H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. The average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. The generation time determined by PLM was 31.1 hr with a SD of 4.7 hrs. Transit times for each stage were: TG1 = 10.6 hr, TS = 9.9 hr, TG2 = 9.9 hr, and TM = 0.7 hr. Repeated experiments using continuous labeling with 3H-TdR demonstrated a TG2 of 6.3 hr. The longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give TG1 = 14.1 hr, TS = 9.9 hr, TG2 = 6.3 hr, and TM = 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. The average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

Establishment and growth kinetics of the mutant Ehrlich-Lettré ascites cell strain HD33 in permanent suspension culture

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 X 10(-5) of the explanted cells continued to grow in vitro. The resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. The corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. The duration of the G1 phase was effectively zero. Both PLM data and [3H]/[14C] thymidine double-labelling measurements revealed an S-phase duration of between 11 and 12 hr. The G2 phase lasted 3-5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

Production of insulin-like growth factor-II (MSA) by endoderm-like cells derived from embryonal carcinoma cells: possible mediator of embryonic cell growth

The present study was carried out to determine if an insulin-like growth factor (IGF) type activity might be produced by embryonal carcinoma-derived cells. The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm-like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid. When growth medium conditioned by Dif 5 cells is chromatographed on Sephadex G-75 in 1 M acetic acid two peaks of activity are observed which compete for specific [125I]iodo multiplication stimulating activity (MSA) binding to PYS cells. MSA is the rat homologue of human IGF-II. The high molecular weight fraction (Mr approximately 60K) apparently corresponds to IGF-binding protein as determined by its ability to bind [125I]iodo-MSA. The low molecular weight fraction (Mr approximately 8K) is biologically active as this fraction stimulates [3H]thymidine incorporation into serum-starved chick embryo fibroblasts. Radioimmunoassay data indicate that the IGF-like activity produced by Dif 5 cells is more closely related to IGF-II than to IGF-I. Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA-like activity, while PYS-2 (parietal endoderm-like) cells produced about 16 ng MSA/10(6) cells/24 hr as determined by radioimmunoassay. Dif 5 and PSA-5E (visceral endoderm-like) cells, are found to secrete significant amounts of MSA into the growth medium (30-50 ng MSA/10(6) cells/24 hr). These findings offer further support to a proposal that MSA (IGF-II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic growth factor.     

Lithium Chloride and Trypan Blue Induce Abnormal Morphogenesis by Suppressing Cell Population Growth

Full primitive streak stage chick embryos were cultured in vitro for 20 hrs and monitored every 4 hr for morphology, cell number and blastoderm area. In normal embryos, the cell population growth is exponential and correlates directly with Increasing morphological rank. The chick blastoderm area expands in two waves, one immediately after gastrulation and another after 16 hr in culture, while cell population growth is predominant between 4–16 hr. Trypan blue and LiCI inhibit cell population growth, epiboly and shaping of organ primordia. Both teratogens induce a similar spectrum of abnormalities although the severity of abnormal development is greater with LiCl for the given dose. In most abnormal embryos the cell population size and blastoderm area are inhibited most, which is detectable already after 12 hr of culture. We have established that the cell population growth, morphogenesis and area expansion constitute a parametric hierarchy with the cell population growth as the most independent parameter in regulating normal morphogenesis.     

Evaluation of methods for determining 6-hydroxydopamine cytotoxicity

Summary The toxic effects of 6-hydroxydopamine on the human neuroblastoma cell line SK-N-SH-SY5Y (SY5Y) and the Chinese hamster ovary (CHO) cell line were measured with five viability assays. Four of the assays (attachment efficiency, plating efficiency, amino acid incorporation into acid-precipitable proteins, and Trypan Blue dye exclusion) showed higher drug susceptibility in SY5Y cells than CHO cells. Only growth inhibition (proliferation index) gave results indicating greater sensitivity in CHO cells. Over a time span of 48 hr, injured cell populations lost vital functions in the following order: attachment ability, amino acid incorporation, proliferative capacity, and dye exclusion. Recovery of each of the functions occurred in sublethally injured populations. Monitoring the extinction and recovery of vital functions permitted the accurate determination of a drug concentration (30 μg/ml) selectively toxic for SY5Y cells. A strong correlation was noted between relative values for amino acid incorporation 3 hr after drug treatment, attachment efficiency at 24 hr, and dye exclusion at 24 and 48 hr. We concluded that Trypan Blue dye exclusion and amino acid incorporation were suitable methods for comparing the effects of cytotoxins on different cell lines, provided they were performed at the appropriate time after treatment with the toxin. The combined techniques yield both population and individual cell data, are simple to do, and are applicable to nondividing cell populations. This work was supported by an NIH National Research Service Award GM07204 to E. T. C., a gift from the Lola-Wright Foundation, NINCDS Grants NS14034 and NS15234, Robert Welch Grant H698, and an RCDA (NS00213) to J. R. P.     

Sequential study on the influence of adriamycin on cell proliferation and ultrastructure of cultured mammary tumor cells

The time-dependent cytocidal and growth inhibitory effects of Adriamycin (ADM) on monolayer cultures of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor cells were analyzed. The inhibitory effect on cell proliferation was assessed by colony formation in soft agar. Growth inhibition and [3H]thymidine labeling indices clearly demonstrate a dose-dependent antimitotic and cytotoxic effect of the drug. At low concentrations (10(-9)-10(-8) M), 90-100% of cells survived 24-hr exposure. At a higher concentration (10(-5) M), 75-80% of cells survived after 8-hr exposure; by 72 hr only 20-30% of the cells remained. Autoradiographic examination of the pulse-labeled cultures demonstrated no change in the proportion of cells in S-phase during the first 4 hr of treatment. Subsequently DNA synthesis was completely abolished and remained inhibited for the duration of the experiment (72 hr). Clonogenic assay revealed a complete arrest of growth in cells exposed to 10(-5) M ADM and greater than 60% inhibition of cell proliferation at 10(-7) M. Ultrastructural changes were not observed in cells during the first 4 hr of treatment; however, after 8 hr most surviving cells exhibited alterations in nuclear chromatin. The surviving cells showed mitochondrial degeneration, myelin body formation, and vacuolization of the endoplasmic reticulum. This study shows the potential usefulness of the primary culture system in drug evaluation. In addition, serial observation of the effects of ADM revealed a cell subpopulation of the primary culture with differential sensitivity to the drug.     

CYTOKINETIC ANALYSIS OF THE JB-1 ASCITES TUMOUR AT DIFFERENT STAGES OF GROWTH

The cell kinetics of the murine JB-1 ascites tumour have been investigated on days 4, 7 and 10 after transplantation of 2·5 × 10⁶ cells. The experimental data, growth curve, percentage of labelled mitoses curves, continuous labelling curves and cytophotometric determination of single-cell DNA content have been analysed by means of a mathematical model for the cell kinetics. The important result was the existence of 8% non-cycling cells with G₂ DNA content in the 10-day tumour, while only 0·2 and 0% were observed in the 7- and 4-day tumours, respectively. The doubling times determined from the growth curve were 22·8, 70 and 240 hr, respectively, in the 4-, 7- and 10-day tumours. Growth fractions of 76, 67 and 44% were calculated for the same tumour ages. The mean cell cycle time increased from 14 to 44 hr from day 4 to 7 due to a proportional increase in the mean transit time of all phases in the cell cycle. In the 10-day tumour, the mean cell cycle changed to 41 hr and T _G1 decreased to 0·5 hr. The cell production rate was 4·3%/hr in the 4-day tumour, 1·2%/hr in the 7-day tumour and 1·0%/hr in the 10-day tumour. The cell loss rates in the same tumours were 1·3, 0·2 and 0·7%/hr, respectively. The analysis made it probable that the mode of cell loss was an age-specific elimination of non-cycling cells with postmitotic DNA content.     

Growth requirements of ferret tracheal epithelial cells in primary culture

In mass cell culture conditions, protease dissociated ferret tracheal epithelial cells (FTE) proliferated in growth factor-supplemented F12 medium to high cell densities (0.5 X 10(5) cells/cm2) with an average population doubling time of 24 hr. The growth factor constituents of the F12 medium included epidermal growth factor (25 ng/ml), insulin (1 microgram/ml), transferrin (10 micrograms/ml), hydrocortisone (18 ng/ml), hypothalamus extract (30-100 micrograms/ml), and conditioned medium from mouse 3T3 fibroblasts. Growth of these cells under clonal conditions was achieved by the partial replacement of F12 medium with M199 medium which was attributed, in part, to the presence of vitamin A in M199 medium. Serum did not stimulate the growth of FTE cells. The epithelial cell nature of these cells in culture was confirmed by ultrastructural features and by immunofluorescent staining for fibronectin.     

Improved electrotransformation frequencies of Corynebacterium glutamicum using cell-surface mutants

The electrotransformation efficiency for homologously- and heterologously-derived plasmid DNA was determined for two families of Corynebacterium glutamicum strains derived from ATCC13059 (AS019 and auxotrophic, cell surface mutants MLB133 and MLB194) and ATCC13032 (parent strain and restriction-minus mutants RM3 and RM4), following their growth in LBG supplemented with glycine plus isonicotinic acid hydrazide (INH). Electrotransformation efficiencies of MLB133 were up to 100-fold higher than for strain ASO19 and, when using heterologously-derived plasmid DNA, MLB133 showed efficiencies comparable to or better than strains RM3 and RM4, demonstrating the relative importance of cell surface structures in impeding DNA uptake in C. glutamicum. Transmission electron microscopy analysis of cell surface structures showed that strain MLB133 has a thin cell wall relative to AS019 and growth in either glycine or INH further diminished its thickness. Both RM3 and RM4 were more sensitive to INH than ATCC13032 and growth in glycine plus INH further improved transformation efficiency. The mycolic acid composition of these strains is described and the impact of glycine and INH on this is reported.