HTC cells

HAU HECS Hep cells HPRT HSV HTC cells IAA ITES haemagglutinin unit human endothelial cell supernatant human epithelial cells hypoxanthine phosphoribosyl transferase herpes simplex virus hepatoma tissue culture cells indole acetic acid medium supplement containing insulin, transferrin, ethanolamine and selenium... 

Baker, M. E., Vaughn, D. A., and Fanestil, D. D., Competitive inhibition of dexamethasone binding to the glucocorticoid receptor in HTC cells by tryptophan methyl ester, /. Steroid Biochem., 13[8], 993, 1980. 

Fig. 9. Relation between partition coefficient. in various solvent systems (abscissa) and permeability coefficients (ordinate). The permeants are numbered as in Fig. 8, and the data refer to the study of Giorgi and Stein [19] on a rat hepatoma cell line (HTC cells) grown in culture. The lower line and filled circles are for decane. Straight lines are drawn through the data, with slopes as indicated.

Fig. 10. Calculated intramembrane diffusion coefficients across membranes of (A) Nil 8 hamster fibroblasts and (B) the HTC rat hepatoma cell line, as a function of molecular weight of the permeant. Ordinate logarithm of membrane thickness, taken as 50-10 cm, multiplied by the permeability coefficient (in cm/sec) and divided by the octanol/water partition coefficient. Abscissa logarithm of molecular weight. Data taken from Giorgi and Stein [19]. Permeants indicated as follows large circle, mean value for all the steroids of Fig. 8 Ur, urea Thw, thiourea Gly, glycerol Ap, antipyrine. Regression lines through the points have slopes of —3.9 for the Nil 8 cells and —3.7 for the HTC cells.

Steinbach OM, Wolterbeek HT. 1993. Effects of zinc on rat hepatoma HTC cells and primary cultured rat hepatocytes. Toxicol AppI Pharmacol 118(2) 245-254. 

Fig. 1. Electron micrograph of an HTC cell. X 9,000. (Photo taken by Dr. Albert Jones, Department of Medicine, University of California, San Francisco).

Using both synchronized and random populations, we have studied the characteristics of the generation cycle of HTC cells (Martin et al., 1969b). By conventional methods, the duration of mitosis was determined to be 0.75 to 1 hour of G1,8 to 9 hours of S, 8 to 9 hours and of G2, 4 to 5 hours. The events occurring in the cell cycle which control the synthesis of TAT will be discussed in greater detail below. 

Fig. 2. Induction of tyrosine aminotransferase in HTC cells by dexamethasone phosphate. A culture of HTC cells, grown to a density of about 8x10 cells/ml, was resuspended in fresh medium and divided into two portions. To the first portion, dexamethasone phosphate was added to a final concentration of 5 X 10-5 M. The other portion was used as a control. Enzyme activity was assayed as described in Martin et al., 1969a, and is expressed as milliunits of enzyme per milligram of cell protein. (From Tomkins et al. Science, 166 1474.)...

Studies on the early kinetics of TAT induction in HTC cells have suggested that following the addition of the inducer there is a lag of 30 minutes to 1 hour before an increase in the rate of enzyme synthesis occurs (Granner et al., 1970). Thereafter, the rate of amino acid incorporation into TAT increases for the next 5 or 6 hours, after which it reaches a steady state, 5- to 15-fold above the basal rate, which is maintained as long as the steroid inducer remains in contact with the cells (Thompson, et al., 1966 Granner et al., 1968b Granner et al., 1970). 

These conclusions are consistent with the less direct observations that cycloheximide, puromycin (Peterkofsky and Tomkins, 1967), and amino acid starvation (Levinson and C. Klein unpublished) can prevent the steroid-mediated induction of TAT in HTC cells. Although such observations, frequently made with inducible eukaryotic systems, are often interpreted to mean that an inducer stimulates the rate of specific enzyme synthesis, it is clear that inhibitor studies indicate only that concomitant protein synthesis is required for induction to occur. Thus, unless specifically shown by rate studies on enzyme synthesis, such indirect data are equally consistent with decreased rates of enzyme degradation (Schimke et al., 1967) or protein synthesis-dependent enzyme modification (Griffin and Cox, 1966). 

THE GENERAL EFFECTS OF STEROID HORMONES ON HTC CELL FUNCTION... 

The biologic activity of the different classes of steroids in HTC cells cannot be explained by differences in their own metabolism, or by their effects on cell growth. 

Fig. 3. Effects of anti-inducers on TAT induction. HTC cell suspensions (600,000 cells/ml) were incubated with either 1, cortisol, 1 x 2, testosterone, 1 x 10-5/1 3,17a-methyl testosterone, 1...

HTC cells do not, however, contain detectable cyclase activity (Granner et al., 1968a), and yet they are stimulated by the glucocorticoids to synthesize TAT. Furthermore, the synthesis of TAT is not affected by the addition to the culture of either cAMP or its dibutyryl derivative (Granner et al., 1968a Stellwagen, Dritz Tomkins, unpublished). Inhibitors of phosphodiesterase which catalyze the hydrolysis of the cyclic nucleotide have no effect on the steroid induction of the enzyme. 

Fig. 4. Effect of inhibitors of macromoiecular synthesis on the degradation of TAT. HTC cells in suspension culture vvere induced with cortisol and labeled for 4 hours with leucine. At zero time the cells were washed free of inducer and leucine and were resuspended in either fresh growth medium (A) or buffered saline (B). (The TAT contained 1,400 cpm per mg protein at zero time.) To one half of the cell suspension in growth medium (A) actinomycin D (AMD) (5/xg/ml) was added at zero time, and aliquots were removed from both cultures for determination of TAT enzymic activity...

In HTC cells, some type of transcriptional control appears to determine whether or not specific genes can be expressed in different parts of the mammalian cell cycle (Martin et al., 1969a Martin and Tomkins, 1970 Tomkins et al., 1969). However, this regulation appears to be inducer-independent. Furthermore, the behavior of inducible enzymes in a number of eukaryotic systems, including HTC cells, has suggested that a second level of regulation of gene expression must also exist. 

Earlier experiments had shown that in HTC cells as well as most other inducible systems concomitant RNA synthesis is required for induction (Thompson et al., 1966 Peterkofsky and Tomkins, 1967). According to Figure 5, when RNA synthesis is blocked, even though R may be neutrahzed by inducer, the concentration of mRNA cannot increase, and, therefore, induction does not occur. 

A number of other experimental results from various eukaryote cells can also be explained by the model shown in Figure 5. An example is illustrated in Figure 6. HTC cells were induced overnight following which the culture was deinduced by removing the inducer. Enzyme synthesis then slowed, as shown by the decreasing concentration of the... 

Fig. 6. Messenger rescue" experiment. The HTC cell suspensions (800,000 cell/ml) were incubated in induction medium with 1 x M cortisol for 17 hours. At that time an 8-ml sample of cell suspension was added to fresh warmed induction medium free of steroid (37 C) and to warmed induction medium containing cortisol (1 x Both suspensions were further incubated at 37°C,...

A more dramatic prediction of the post-transcriptional control model is also substantiated in Figure 6. Following deinduction of a culture inhibition of RNA synthesis should rescue the repressed mRNA and reactivate enzyme formation. Experiments in accord with this prediction were done in HTC cells (Tomkins et al., 1969,1970 Levinson et al., unpublished), and in cultured fetal hamster cells (Bausserman and Nebert, 1970). The results are in remarkably good agreement. In the experiment using HTC cells (Fig. 6) the inducer was removed from the induced culture, and after specified intervals AMD was added to the deinduced culture. As illustrated, TAT formation was reactivated shortly after addition of the inhibitor. Similar results are obtained when the nucleoside analog mercaptopyridethylbenzimidazole (MPB) was used instead of actinomycin D (Levinson, unpublished Tomkins et al., 1970). 

Fig. 7. TAT activity during mitosis and G1. Monolayer cultures of HTC cells were induced with dexamethasone phosphate (Dex), 10-6 M, for 24 hours prior to exposure to 2 x 10-7/1 colcemid. After 12 hours of colcemid treatment, those cells arrested in metaphase were selectively harvested. The adherent interphase cells served as a control population in the experiment in part A. The populations of synchronized cells (mitotic indices greater than 90 percent) were washed by centrifugation (200 x g) at 37°C and resuspended in fresh, prewarmed medium containing neomycin (50 jug/ml) and, when indicated, Dex 10-5 M. These suspension cultures (about 500,000 cells/ml) were maintained at 37°C, and the appropriate drugs were added as Indicated. Aliquots were removed at the intervals indicated and chilled. The cells were washed with neutral buffer prior to being quick-frozen and stored for subsequent determinations. TAT-specIfic activities (nmoles of product/min/mg protein) were normalized to an initial value of 100 because, even though the induction in each case was at least eightfold, the maximally induced levels varied from experiment to experiment. In the experiment depicted in A, the population of synchronized cells was washed free of colcemid at zero time. Dex was also removed from one half of the population (0—0), but in the other half Dex (10-5 M) was present...

An important question is, of course, the precise mechanism of steroid action. Two apparently inconsistent facts must be resolved. The first is the nuclear localization of inducing steroids in HTC cells (Baxter and Tomkins, 1970) as well as in most other steroid-sensitive systems (see Tomkins and Martin, 1970, for references). The second is the evidence that R functions at the site of protein synthesis in the cytoplasm. If the hormones do indeed modulate R function, they must do so by an as yet unknown indirect mechanism. 

Granner, D. K., E. B. Thompson, and G. M. Tomkins. 1970. Dexamethasone phosphate induced synthesis of tyrosine aminotransferase in HTC cells. J. Biol. Chem., 245 1472. 

Martin, D. W., Jr., and G. M. Tomkins. 1970. The appearance and disappearance of the post-transcriptional repressor of tyrosine aminotransferase synthesis during the HTC cell cycle. Proc. Nat. Acad. Sci. U. S. A., 65 1069. 

In these coupled reactions, the overall transformation of glucose to fructose is accompanied by a transfer of hydrogen from NADPH to NAD. Studies on the activity of aldose reductase and sorbitol dehydrogenase as well as the presence of sorbitol in seminal vesicles of several species and rat coagulating gland are consistent with the operation of the sorbitol pathway as a major determinant of seminal fructose biosynthesis (248, 252-260). Nonphosphorylative conversion of glucose to sorbitol has also been demonstrated in placenta (258, 261, 262), smooth muscle of the aorta (263), the lens of the eye (264, 265), erythrocytes (266) and hepatoma-derived (HTC) cells in culture (267), and possibly normal liver cells (267, 268). 

M39 Mitchell, A. D. and Hoogenraad, N. J. De novo pyrimidine nucleotide biosynthesis in synchronized rat hepatoma (HTC) cells and mouse embryo fibroblast (3T3) cells. Exp. Cell Res., 93, 105-110 (1975)... 

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