Tumour cell growth in culture: dependence on arginine

2004 ◽  
Vol 107 (4) ◽  
pp. 371-379 ◽  
Author(s):  
Giuseppe CASO ◽  
Margaret A. McNURLAN ◽  
Nelson D. McMILLAN ◽  
Oleg EREMIN ◽  
Peter J. GARLICK
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Tumour Cell ◽  
Urea Cycle ◽  
Breast Tumour ◽  
Cell Protein ◽  
Tumour Cell Line ◽  
Intracellular Enzyme ◽  
Polyamine Synthesis ◽  
Mcf 7

The amino acid arginine has been shown to affect the growth of several tumours, although the mechanisms of its action are not clear. In the present study, using a human breast tumour cell line (MCF-7), we investigated the arginine requirements of tumour cells for optimal protein synthesis and growth, and the metabolic pathway responsible for the arginine-dependent growth. The results showed that MCF-7 cells are highly dependent on arginine for growth and that the requirement for arginine is much higher than for an indispensable amino acid, leucine, indicating that arginine is needed for pathways other than protein synthesis. In arginine-free cultures, growth could be completely restored by the urea cycle intermediate citrulline. However, arginine could not be replaced by the urea cycle intermediate and the direct precursor for polyamine synthesis, ornithine, or by the polyamine putrescine, suggesting that the high dependence on arginine is not due to a requirement for polyamine synthesis. Moreover, inhibition of NOS [NO (nitric oxide) synthase] did not affect cell protein synthesis and growth, and the arginine analogue and substrate for NOS, homoarginine, could not replace arginine, implying that the conversion of arginine into NO is not involved in the growth-promoting effects of arginine. The major determinant for the high dependence of MCF-7 cells for arginine was found to be the irreversible conversion of this amino acid into ornithine by the intracellular enzyme arginase. The conversion into ornithine caused a progressive depletion of arginine from the culture medium, which ultimately inhibited cell protein synthesis and halted growth. Intracellular arginase activity may be the major factor determining the requirement for arginine of all cells in culture.

scholarly journals Synthesis of recombinant human procollagen II in a stably transfected tumour cell line (HT1080)

1994 ◽  
Vol 298 (1) ◽  
pp. 31-37 ◽  
Author(s):  
A Fertala ◽  
A L Sieron ◽  
A Ganguly ◽  
S W Li ◽  
L Ala-Kokko ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Tumour Cell ◽  
Copy Number ◽  
Gene Copy Number ◽  
Basement Membranes ◽  
Gene Copy ◽  
Nucleotide Sequencing ◽  
Tumour Cell Line ◽  
Col2a1 Gene

Apparently because the biosynthetic pathways involve eight or more highly specific post-translational enzymes, it has been difficult to obtain expression of genes for fibrillar collagens in recombinant systems. Here two constructs of the human gene for procollagen II (COL2A1) were prepared, one with about 0.5 kb of a promoter for a procollagen I gene (COL1A1) and the other with about 4 kb of the promoter for the procollagen II gene. The constructs, together with a neomycin-resistant gene, were transfected into a human tumour cell line (HT1080) that synthesizes the collagen IV found in basement membranes, but does not synthesize any fibrillar collagen. About two per 100 clones resistant to the neomycin analogue G418 synthesized and secreted human procollagen II. Milligram quantities of the recombinant procollagen II were readily isolated from the cultured medium. The recombinant procollagen II had the expected amino acid sequence as defined by nucleotide sequencing of mRNA-derived cDNA and the expected amino acid composition as defined by analysis of procollagen II that was converted into collagen II by digestion with procollagen N- and C-proteinases. Also, analysis of the carbohydrate content indicated that there was glycosylation of some of the hydroxylysine residues but no evidence of post-translational overmodification of the residues. In addition, the protein was shown to have a native conformation as assayed by a series of protease digestions. No essential differences were found between clones transfected with the COL2A1 gene construct containing the COL1A1 promoter and the similar construct containing the COL2A1 promoter in terms of number of clones synthesizing recombinant procollagen II and the levels of expression. With both constructs, the expression of the COL2A1 gene was closely related to copy number. The results demonstrated therefore that it is not essential to use a promoter for a gene normally expressed in a host cell in order to obtain gene copy-number-dependent expression of an exogenous collagen gene in stably transfected cells.

scholarly journals The mysteries of nitrogen balance

1999 ◽  
Vol 12 (1) ◽  
pp. 25-54 ◽  
Author(s):  
J. C Waterlow
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Urea Cycle ◽  
N Balance ◽  
Single Amino Acid ◽  
Whole Body ◽  
Acid Oxidation ◽  
Physiological Level ◽  
Body Protein ◽  
Urea Production

AbstractThe first part of this review is concerned with the balance between N input and output as urinary urea. I start with some observations on classical biochemical studies of the operation of the urea cycle. According to Krebs, the cycle is instantaneous and automatic, as a result of the irreversibility of the first enzyme, carbamoyl-phosphate synthetase 1 (EC 6.3.5.5; CPS-I), and it should be able to handle many times the normal input to the cycle. It is now generally agreed that acetyl glutamate is a necessary co-factor for CPS-1, but not a regulator. There is abundant evidence that changes in dietary protein supply induce coordinated changes in the amounts of all five urea-cycle enzymes. How this coordination is achieved, and why it should be necessary in view of the properties of the cycle mentioned above, is unknown. At the physiological level it is not clear how a change in protein intake is translated into a change of urea cycle activity. It is very unlikely that the signal is an alteration in the plasma concentration either of total amino-N or of any single amino acid. The immediate substrates of the urea cycle are NH3 and aspartate, but there have been no measurements of their concentration in the liver in relation to urea production. Measurements of urea kinetics have shown that in many cases urea production exceeds N intake, and it is only through transfer of some of the urea produced to the colon, where it is hydrolysed to NH3, that it is possible to achieve N balance. It is beginning to look as if this process is regulated, possibly through the operation of recently discovered urea transporters in the kidney and colon. The second part of the review deals with the synthesis and breakdown of protein. The evidence on whole-body protein turnover under a variety of conditions strongly suggests that the components of turnover, including amino acid oxidation, are influenced and perhaps regulated by amino acid supply or amino acid concentration, with insulin playing an important but secondary role. Molecular biology has provided a great deal of information about the complex processes of protein synthesis and breakdown, but so far has nothing to say about how they are coordinated so that in the steady state they are equal. A simple hypothesis is proposed to fill this gap, based on the self-evident fact that for two processes to be coordinated they must have some factor in common. This common factor is the amino acid pool, which provides the substrates for synthesis and represents the products of breakdown. The review concludes that although the achievement and maintenance of N balance is a fact of life that we tend to take for granted, there are many features of it that are not understood, principally the control of urea production and excretion to match the intake, and the coordination of protein synthesis and breakdown to maintain a relatively constant lean body mass.

scholarly journals Unoccupied binding sites for oestrogen in nuclei of a breast tumour cell line (MCF-7)

1981 ◽  
Vol 200 (3) ◽  
pp. 515-520 ◽  
Author(s):  
A Geier ◽  
M Haimsohn ◽  
Z Malik ◽  
B Lunenfeld
Keyword(s):  
Nuclear Receptor ◽  
Fold Increase ◽  
Breast Tumour ◽  
Considerable Change ◽  
Tumour Cell Line ◽  
Nuclear Fraction ◽  
Oestrogen Receptors ◽  
Triton X ◽  
Mcf 7 ◽  
In Cells

1. A method to measure both occupied and unoccupied oestrogen receptors directly in the crude nuclear fraction of the MCF-7 cells was developed. The receptors had high affinity for oestradiol (Kd approx. 0.7 nM) and binding specificity characteristics of oestrogen receptors. 2. A substantial amount of the unoccupied receptors were found in the crude nuclear fraction. 3. Several experiments excluded the possibility that the unoccupied nuclear receptor might be a cytoplasmic contaminant. (a) Multiple extractions with Tris buffer released about 75% of the total receptor content, leaving the rest unextractable in the crude nuclear fraction. (b) Nuclei purified by centrifugation through 1.8M-sucrose and treatment with 0.7% Triton X-100, or by centrifugation through 50% glycerol with 0.1% Triton X-100 contained similar amounts of unoccupied receptors to that found in the crude nuclear fraction. (c) In cells cultured during 5 days after preconfluency a 3-fold increase in the amount of unoccupied cytoplasmic receptors occurred, whereas the amount of unoccupied nuclear receptors did not change significantly and conversely in cells exposed to increasing concentrations of oestradiol the unoccupied cytoplasmic receptor was continuously depleted but no considerable change in the unoccupied nuclear receptor was found.

scholarly journals Prolyl-tRNA-based rates of protein and collagen synthesis in human lung fibroblasts

1981 ◽  
Vol 198 (2) ◽  
pp. 249-258 ◽  
Author(s):  
J N Hildebran ◽  
J Airhart ◽  
W S Stirewalt ◽  
R B Low
Keyword(s):  
Protein Synthesis ◽  
Steady State ◽  
Amino Acid ◽  
Collagen Synthesis ◽  
Human Lung ◽  
Specific Radioactivity ◽  
Cell Protein ◽  
Free Proline ◽  
Wide Range ◽  
The Relationship

Knowledge of the dynamics of collagen turnover requires information regarding rates of synthesis of this group of connective-tissue proteins. The relationship of various amino acid pools to the tRNA precursor pool used for protein synthesis is known to vary between different cell types and tissues, even for essential amino acids. We studied extracellular, intracellular and tRNA-proline pools in cultured human lung IMR-90 fibroblasts to determine the relationship between them as candidate proline precursor pools for total protein and collagen synthesis. Time-course experiments showed that the three proline pools attained distinctly different steady-state specific radioactivities (extracellular greater than intracellular greater than tRNA) at the extracellular proline concentration of 0.2 mM. The kinetics of radioisotope incorporation into cell protein and collagenase-digestible protein indicated that the intracellular free proline pool could not be used reliably as a precursor for calculating synthetic rates. However, tRNA-proline behaved isotopically as if it were the precursor and provided synthesis rates 2-3-fold higher than those calculated by using either free proline pool. The incorporation of labelled lysine and leucine was constant over a wide range of extracellular proline concentrations. Fractional rates of protein synthesis based on tRNA-amino acid were the same with [3H]phenylalanine as with [3H]proline. The specific radioactivity of cell-associated hydroxyproline reached a steady-state value 8-10h after radioisotope administration which matched the mean tRNA-proline specific radioactivity, suggesting that tRNA-proline is not isotopically compartmentalized. A model of cellular proline-pool relationship is presented and discussed.

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