Pirkko Pohjanpelto

Deprivation of a single amino acid induces protein synthesis-dependent increases in c-jun, c-myc, and ornithine decarboxylase mRNAs in Chinese hamster ovary cells.

Genes of higher eucaryotic cells are considered to show only a limited response to nutritional stress. Here we show, however, that omission of a single essential amino acid from the medium caused a marked rise in the mRNA levels of c-myc, c-jun, junB and c-fos oncogenes and ornithine decarboxylase (ODC) in CHO cells. There was no general accumulation of mRNAs in amino acid-starved cells, since the gamma-actin, beta-tubulin, protein kinase C, RNA polymerase II, and glyceraldehyde-3-phosphate dehydrogenase mRNAs and the total poly(A)+ mRNA were not increased. The levels of c-myc, ODC, and c-jun mRNAs were elevated more by amino acid starvation than by inhibition of protein synthesis with cycloheximide, which is known to increase the levels of these mRNAs. Importantly, however, cycloheximide present during amino acid starvation reduced the rise in the levels of the mRNAs down to the level obtained with cycloheximide alone. This implies that protein synthesis is required for the accumulation of c-myc, ODC, and c-jun mRNAs in amino acid-deprived cells. The junB and c-fos mRNAs, instead, were increased to the same extent or less by amino acid starvation than by cycloheximide treatment. The accumulation of the c-myc mRNA in amino acid-starved cells was due to both stabilization of the mRNA and increase of its transcription. The rise in the c-jun mRNA level seemed to be caused merely by stabilization of the mRNA. Further, despite the inhibition of general protein synthesis, amino acid starvation led to an increase in the synthesis of c-myc polypeptide. The results suggest that mammalian cells have a specific mechanism for registering shortages of amino acids in order to make adjustments compatible with cellular growth.

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine-and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.

Dissociation of the early antiproliferative action of methylglyoxal bis(guanylhydrazone) from polyamine depletion: A comparison of the effects of DL-α-difluoromethyl ornithine and methylglyoxal bis(guanylhydrazone) on the growth of human fibroblasts

Association of polyamines with cell growth appears to be a general phenomenon [l-3] . The mechanism of action of these compounds is, however, still largely obscure. This problem has been recently approached by attempts to block polyamine synthesis wth various inhibitors [3 ] . Methylglyoxal bis(guanylhydrazone) (MGBG) which prevents the synthesis of spermidme and spermme by blockmg the decarboltylation of adenosylmethionine [4] retarded DNA synthesis in concanavahn A-activated bovine lymphocytes at low concentrations sufficient to prevent polyamine synthesis but did not affect RNA or protein synthesis [5] . InhIbition of DNA synthesis followmg exposure to MGBG has also been observed in cultures of human fibroblasts WI-38 [6] and HeLa cells [7]. In addition, rat embryo fibroblasts [8] and rat brain tumor cells [9] were reportedly arrested in G1 after the inhibitor treatment. The results obtamed with this mhibitor, however, should be interpreted with certam reservations, smce m leukemia L 1210 cells the drug has been reported to cause early cytotoxic effects which may be unrelated to Its effects on polyamme metabolistn [lo]. A newly Qscovered irreversible inhibitor of ornithme decarboxylase, DLc+difluoromethyl ormthme (DFMO), prevented the accumulation of putrescme and spermidine 111 rat hepatoma cells, mouse leukemia cells and human prostate adenoma cells, but the growth was not retarded until after a delay of one cell generation [ 1 I] . The present study compares the temporal sequence of polyamine depletion and inhibition of macromolecular synthesis following ad&tion of MGBG or DFMO to cultured human embryomc fibroblasts. The results show that the prevention of polyamme accumulation of DFMO during the first day &d not unmediately affect the rate of DNA synthesis. In contrast, MGBG, at PM levels, caused a marked mhlbltion of the synthesis of DNA and protem as early as 12 h after addition of the drug.’ At that time the accumulation of polyammes proceeded as m the absence of the inhibitor. The initial inhibition of the growth of human fibroblasts by MGBG thus may not be primarily mediated by polyamine metabohsm.

Response of Enteroviruses to Cystine.

Abstract Polioviruses types 1, 2, and 3 were stabilized against heat inactivation by in vitro exposure to L-cystine, 50 μg/ml. The rate of stabilization was fastest with type 1 and slowest with type 3. Strains of the same poliotype may show different rates of stabilization. Coxsackie A9, Coxsackie B3, and ECHO 1 were stabilized by L-cystine at 500–2500 μg/ml. ECHO 3, 6, and 19 showed only slight stabilization, if any.

Polyamine starvation causes parallel increase in nuclear and chromosomal aberrations in a polyamine-dependent strain of CHO.

Deprivation of polyamines and ornithine causes in a polyamine-dependent CHO strain aneuploidy and alterations in nuclear morphology including micronuclei, macronuclei, framented and bulged nuclei. There is also formation of multinucleate cells. The number of micronuclei and certain other nuclear aberrations increase concomitantly with chromosome abberrations.

Relationship between putrescine and the proliferation of human fibroblasts in vitro

Abstract When cells were grown at various temperatures, serum concentrations and cell densities, the conditioned medium from those cultures which showed the largest absolute increase in cell number, had a growth-stimulating effect at the highest dilutions. The growth-stimulating effect of pure putrescine was concentration dependent, and the optimal concentration, causing maximal stimulation of cell proliferation, was highest for those cultures that, without putrescine, showed most cell proliferation.

Two different thermostable variants of poliovirus.

Abstract In a cystineless medium, a thermostable variant was isolated which showed high thermal stability without cystine treatment. Resistance to heat inactivation was increased further by incubation with cystine as well as by removal of oxygen during heating. Reducing agents did not abolish this thermal stability. Another thermostable variant, which was isolated in a cystine-containing medium, expressed its increased thermal stability only after incubation with cystine. The plaques produced by the former variant were smaller than those by the latter.

Arginase activity of different cells in tissue culture

Arginase activity was measured in ten different cell lines and all of them showed arginase activity. However, the amount of the activity in the cells varied more than 1000-fold. The cell density did not appear to affect the arginase activity much, since cultures of human fibroblasts grown to different cell densities exhibited only small variations in arginase activity. Arginase activity was in general higher than that of ornithine decarboxylase and there was no correlation between the two activities. When human fibroblasts were stimulated to proliferate in serum-free medium by adding certain growth factors the activity of ornithine decarboxylase increased markedly prior to the DNA synthesis, while the arginase activity increased more slowly and to a much smaller degree. Two cell lines, baby hamster kidney and mouse 3T3 cells, had low arginase activity when adapted to grow in serum-free medium, but in spite of this they were able to grow in serum-free medium without exogenous ornithine.

Polyamine depletion of cells reduces the infectivity of Herpes simplex virus but not the infectivity of sindbis virus

The effect of polyamines on the viral growth was examined using cell strains that could be effectively depleted of polyamines. In order to avoid the polyamines present in serum we used a polyamine auxotrophic Chinese hamster ovary cell line P22 growing in serum-free medium and Vero cells growing in low serum medium. The final yield of an enveloped RNA virus, Sindbis, in P22 cells was not decreased by depletion of cellular polyamines although the onset of the viral replication was delayed. In contrast the final yield of an enveloped DNA virus, Herpes simplex virus (HSV), was considerably reduced in Vero cells, depleted of polyamines by alpha-difluoromethylornithine, an inhibitor of polyamine synthesis. However, the number of HSV particles detected by electronmicroscopy was not decreased. Southern blot analysis of HSV-DNA from the polyamine depleted and the control cells showed changes in the relative abundance of the DNA fragments suggesting that impairment in DNA synthesis may have caused the decreased infectivity of HSV.

Cycloheximide elicits in human fibroblasts a response characteristic for initiation of cell proliferation

Abstract Earlier I found that a variety of stimuli to proliferation of cultured human fibroblasts caused an increase in the rate of putrescine transport into the cells. This paper reports the effects of cycloheximide on putrescine transport in stationary and growing cultures. Cycloheximide in concentrations that inhibited protein synthesis caused increased putrescine transport in serumstarved and density-inhibited cultures. Similar effects were found with pactamycin, also an inhibitor of protein synthesis. Actinomycin D in concentrations that suppressed messenger RNA (mRNA) synthesis, did not cause increased putrescine transport. When both serum and cycloheximide were added to serum-starved cultures, the increase in putrescine transport was greater than when serum alone was added. However, cycloheximide had an inhibitory effect when added 1–2 h after addition of serum. These results suggest that one or more rapidly metabolizing proteins may be important in the regulation of putrescine transport and initiation of cell growth.