Harry Eagle

Measurement of Cell Growth in Tissue Culture with a Phenol Reagent (Folin-Ciocalteau):

Summary An analytical method for the measurement of cell growth in tissue culture is described, based on the Lowry method for the determination of protein, and employing a phenol reagent (Folin-Ciocalteau) for color development. The results, referred to a bovine serum albumin standard, may be converted to dry weight, nitrogen or cell count by appropriate conversion factors. Those factors are here given for 7 human cell lines and 1 mouse line.

Propagation in a Fluid Medium of a Human Epidermoid Carcinoma, Strain KB

Summary An epidermoid carcinoma of the floor of the mouth was cultivated directly onto a glass surface in a medium consisting of 13 amino acids, 7 vitamins, glucose, salts, and 10% human serum. All of the defined components of this medium had previously been shown to be essential for the propagation of the HeLa cell, and all were used at concentrations permitting maximal growth of that cell. The new line (KB) has been grown continuously in this medium for 5 months, and maintains a generation time in the logarithmic phase of growth of approximately 30 hours.

Requirements for Growth of Single Human Cells "Nonessential" amino acids, notably serine, are necessary and sufficient nutritional supplements

A minimal growth medium supplemented with dialyzed serum, which sufficed for the propagation of a wide variety of human cell strains in heavily inoculated monolayer and suspension cultures, did not permit the regular or optimal growth of small numbers of HeLa, HeLa S3, conjunctiva, or KB cells deriving from suspension cultures. At threshold concentrations of serum, the plating efficiency of single cells was greatly reduced as compared with their plating efficiency in a medium containing dialyzed serum instead of whole serum, and the clones which did develop grew at a slower rate. The nutritional deficiency could be overcome by adding the seven amino acids which are ordinarily not nutritionally essential. In most of the experiments serine alone sufficed.

Amino acid requirements of normal and malignant human cells in tissue culture.

Abstract Three tissue culture cell lines deriving from normal human tissue (liver, conjunctiva, and intestine) and two lines deriving from human cancer [KB (nasopharynx), and J-111 (monocytic leukemia)] have been examined with respect to their amino acid requirements in comparison with those of a HeLa cell and a mouse fibroblast. With the possible exception of tryptophan, all seven cell lines required the same amino acids (arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tyrosine, valine); and in the absence of any one of these 12, cytopathogenic changes developed which culminated in the death of the cell. The provision of non-essential amino acids, purines, pyrimidines, and NH 4 + had a glutamine-sparing effect, but did not eliminate the need for this amino acid. Extremely high and nonphysiological concentrations of glutamic acid (20 mM ) did, however, substitute for glutamine. It is not yet clear whether tryptophan is similarly essential for survival and growth, or whether it is merely growth stimulatory for some of the five cell lines here studied. The concentration of the individual amino acids necessary for optimum growth varied somewhat among the six strains; but there were not significant or consistent differences in this respect between the lines deriving from normal and from malignant tissues.

Glucose and glutamine in poliovirus production by HeLa cells

Abstract In monolayers of HeLa cells, glucose, glutamine, and balanced salts promoted essentially maximal yields of poliovirus. Fructose substituted for glucose in this respect, but not galactose, ribose, ribose plus pyruvate, or ribose-5PO 4 . The effective concentrations of glucose and fructose for viral synthesis were the same as those required for cell growth. Glutamic acid at high concentrations (10–20 mM) substituted for glutamine in poliovirus production. The effective concentrations of glutamic acid and glutamine were approximately the same as those required for cell growth. Adaptation of the cells to glutamic acid reduced the concentration of glutamic acid necessary for virus production. The need for added glutamine in order to obtain maximal virus production is probably related to its rapid disappearance from the free amino acid pool.

Utilization of Dipeptides by Mammalian Cells in Tissue Culture

Summary Twelve amino acids had proved essential for the survival and growth of both the mouse fibroblast (strain L) and a human carcinoma cell (strain HeLa). In media lacking a single essential amino acid; in which the cells would otherwise degenerate and die, normal growth and multiplication were obtained on the addition of a dipeptide containing the essential amino acid. The effective concentrations of the dipeptide were of the same order of magnitude as those required of the simple amino acid. It remains to be seen whether the dipeptide is incorporated into cellular protein as such, or whether it is first split into its component amino acids(3).

Viral susceptibility of a human carcinoma cell (strain KB).

Summary 1) A human epithelioid carcinoma cell in tissue culture (strain KB) has been found susceptible to a number of viruses, including poliomyelitis, herpes simplex, vaccinia, a number of APC strains, LCM and EMC. Marked cytopathogenic effects were produced in 1-7 days, and viral multiplication was shown by complement fixation, tissue culture, or animal titration of the second passage material. 2) Only partial and irregular cytopathogenic effects were obtained with mumps, yellow fever, and the SVi strain of APC virus, but with apparently continuing elaboration of virus on repeated passage. Both an egg-adapted and a mouse strain of mumps virus were found to have lost their in-fectivity for eggs after several culture passages. 3) Coxsackie virus (strain A9), influenza type B, one APC type, and GD VII had no cytopathogenic effect on this cell, and there was no evidence of viral multiplication. Rabies virus was cytopathogenic in the first passage only; and the harvest of the second passage was not infectious on intracerebral inoculation in mice.

Relative growth-promoting activity in tissue culture of co-factors and the parent vitamins.

Summary A number of the co-factors here tested (FMN, DPN, cocarboxylase) had essentially the same activity as the corresponding vitamin (riboflavin, nicotinamide and thiamine, respectively) in promoting the growth of the mouse fibroblast, in terms of either the effective concentration or the amount of growth obtained. Several vitamin conjugates (FAD, TPN, coenzyme A and pyridoxal phosphate) proved significantly less active than the parent vitamin (riboflavin, nicotinamide, pantothenate, and pyridoxal, respectively). The present experiments provide no information as to the degree to which these differences may reflect only variations in cell permeability, rather than partial blocks in the utilization of these specific co-factors, or in their conversion to a more active form. Natural citrovorum factor, however, was somewhat more active than folic acid. Of the vitamin congeners or precursors here tested, pyridoxine and pyridoxal were essentially equivalent in activity; nicotinic acid was significantly less active than either nicotinamide or DPN in terms of the amount of growth obtained, although the effective concentrations were of the same order of magnitude; while PABA was wholly inactive as a substitute for folic acid. As with the co-factors, the present experiments do not exclude differential cell permeability as the basis of these differences.

The biosynthesis of poliovirus in cell cultures.

Publisher Summary This chapter considers the recent findings of poliovirus with respect to its biosynthesis. Results of quantitative significance concerning the biosynthesis of poliovirus have come almost exclusively from the use of dispersed mammalian cells, either in primary culture, exemplified by monkey kidney epithelium or in serially propagated cultures, such as the HeLa cell. Either of these cell lines can be used for both the production of virus and its quantitative plaque assay. The particular value of poliovirus is that it is an RNA-containing virus, the replication of which can be studied under experimental conditions approximating those of the coliphage system. The detailed study of the chemical and biophysical properties of the virus particle promises to be rewarding in itself. There is preliminary evidence for free sulfhydryl groups in the protein coat of poliovirus. This should allow attachment of heavy atoms such as mercury to yield a product that would be of aid to the X-ray crystallographer in studies on the structure of the particles.

Effect of Prior Immunization on Bactericidal Action of Penicillin in vivo

Summary and Conclusions In the group A streptococcal infection of mice here studied, and under the conditions of the present experiments, the presence of a significant degree of immunity had no effect on the direct bactericidal action of penicillin. This proceeded at the same rate in the immunized animals as in control mice simultaneously inoculated, and continued for the same period of time. The difference in the 2 groups lay in the course of the infection after penicillin had fallen to ineffective levels. In non-immune mice, the surviving bacteria regularly grew out to cause a fatal infection. In the partially immunized animals, however, a small fraction of the normally curative dose of penicillin sufficed to reduce the bacterial population to levels which could be handled by the host defenses. Those surviving bacteria gradually disappeared in the course of 24-48 hours, after penicillin itself was no longer operative, and the animals survived. Under the conditions of this experiment, the host defenses and the bactericidal action of penicillin proceeded independently of each other, but were mutually supplementary in effecting cure in the partially immunized animals. The choice of a group A streptococcal infection was perhaps unfortunate. The resistance to reinfection in the immunized animals probably reflects enhanced phagocytosis; and in the present experiments, there was no pronounced cellular infiltration at the focus of infection at the time of treatment with penicillin. It would be of interest to carry out similar studies under conditions closer to those usually operative in the natural infection of man, with the cellular host defenses already mobilized at the focus of infection. It would be of interest also to determine, in infections caused by organisms susceptible to the bactericidal action of antibodies and complement, whether the presence of such antibodies modifies the therapeutic action of penicillin and other antibiotics.