Effect of inhibitors of S-adenosylmethionine decarboxylase on the contents of ornithine decarboxylase and S-adenosylmethionine decarboxylase in L1210 cells.

Treatment of L1210 cells with either of two inhibitors of S-adenosylmethionine decarboxylase (AdoMetDC), namely 5'-deoxy-5'-[N-methyl-N-[2-(amino-oxy)ethyl])aminoadenosine or 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine, produced a large increase in the amount of ornithine decarboxylase (ODC) protein. The increased enzyme content was due to a decreased rate of degradation of the protein and to an increased rate of synthesis, but there was no change in its mRNA content. The inhibitors led to a substantial decline in the amounts of intracellular spermidine and spermine, but to a big increase in the amount of putrescine. These results indicate that the content of ODC is negatively regulated by spermidine and spermine at the levels of protein translation and turnover, but that putrescine is much less effective in bringing about this repression. Addition of either spermidine or spermine to the cells treated with the AdoMetDC inhibitors led to a decrease in ODC activity, indicating that either polyamine can bring about this effect, but spermidine produced effects at concentrations similar to those found in the control cells and appears to be the physiologically important regulator. The content of AdoMetDC protein (measured by radioimmunoassay) was also increased by these inhibitors, and a small increase in its mRNA content was observed, but this was insufficient to account for the increase in protein. A substantial stabilization of AdoMetDC occurred in these cells, contributing to the increased enzyme content, but an increase in the rate of translation cannot be ruled out.     

Growth inhibition by methionine analog inhibitors of S-adenosylmethionine biosynthesis in the absence of polyamine depletion

Four methionine analog inhibitors of methionine adenosyltransferase, the enzyme which catalyzes S-adenosylmethionine biosynthesis, were tested in cultured L1210 cells for their effects on cell growth, leucine incorporation, S-adenosylmethionine (AdoMet) formation and polyamine biosynthesis. The IC50 values were as follows: selenomethionine, 0.13 mM; L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cis-AMB), 0.4 mM; cycloleucine, 5 mM and 2-aminobicyclo[2.1.1]hexane-2-carboxylic acid, 5 mM. At IC50 levels, the analogs significantly reduced AdoMet pools by approximately 50% while not similarly affecting leucine incorporation or polyamine biosynthesis. In combination with inhibitors of polyamine biosynthesis, growth inhibition was greatly increased with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase, but only slightly increased with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Overall, the data indicate that the methionine analogs, and particularly L-cis-AMB, seem to inhibit cell growth by interference with AdoMet biosynthesis. Since polyamine biosynthesis is not affected, the antiproliferative effect may be mediated through perturbations of certain transmethylation reactions.     

Adenovirus-mediated expression of both antisense ornithine decarboxylase and S-adenosylmethionine decarboxylase inhibits lung cancer cell growth

Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC). Antisense sequences of ODC and AdoMetDC genes were cloned into an adenoviral vector (named Ad-ODC-AdoMetDCas). To evaluate the effects of recombinant adenovirus Ad-ODC-AdoMetDCas that can simultaneously express both antisense ODC and AdoMetDC, the human lung cancer cell line A-549 was infected with Ad-ODC-AdoMetDCas or the control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were carried out to assess the properties of tumor growth and invasiveness. Our study showed that adenovirus-mediated antisense ODC and AdoMetDC expression inhibits tumor cell growth through blocking the polyamine synthesis pathway. Tumor cells were arrested at the G_1 phase after gene transfer and the invasiveness was reduced. It suggested that the recombinant adenovirus Ad-ODC-AdoMetDCas might be a new anticancer reagent in the treatment of lung cancers.     

Treatment with alpha-difluoromethylornithine plus a spermidine analog leads to spermine depletion and growth inhibition in cultured L1210 leukemia cells

Of the three biological polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), the relevance of Spm to cell proliferation has yet to be defined because of our general inability to deplete it selectively in intact cells. In the present study, Spm depletion was accomplished by treating cultured L1210 cells for 96 hr with alpha-difluoromethylornithine (DFMO) and an analog of Spd such as aminopropylcadaverine, N4-methylSpd, N4-ethylSpd, or homoSpd. DFMO, a specific inhibitor of ornithine decarboxylase, halts continued polyamine biosynthesis and the Spd analog serves as a functional substitute for Spd. Thus, while the Spd analog fulfills the role(s) of Spd in cell proliferation, Spm becomes steadily depleted. In cells treated with DFMO plus the analog, aminopropylcadaverine, Spm pools decline steadily and growth inhibition occus after 48 hr (when Spm pools decline to 60% of control). By 96 hr, Spm is approximately 15% of control and growth is less than 30%. Prevention studies with exogenous polyamines confirm a causal relationship between Spm depletion and growth inhibition. The critical levels of polyamines for cell proliferation to take place were found to be 30% of control for Spd and 60% for Spm. The use of DFMO plus a Spd analog is proposed as a system for studying the cellular consequences of Spm depletion. Spd depletion can be achieved for comparison purposes by treating cells with DFMO alone.     

Characterization of cultured tobacco cell lines resistant to ethionine, a methionine analog

Two cultured tobacco cell lines (Nicotiana tabacum L. cv Xanthi) were selected for resistance to growth inhibition by the methionine analog ethionine. Comparison of the free amino acid pool levels in these lines with those of the ethionine-sensitive parental line showed substantial accumulation of methionine (110×), threonine (18×), and lysine (5×). In vitro enzymic analysis of lysine-sensitive aspartate kinase activity showed the resistant lines to contain 16 times that found in the sensitive line. The lysine-sensitive enzymes from both resistant and sensitive lines coeluted from DEAE-cellulose and exhibited similar K_m values. Both showed identical lysine plus S-adenosylmethionine inhibition profiles suggesting that the elevated activity in the resistant lines is not due to a structural change in the lysine-sensitive enzyme but possibly to the level of its expression.     

An adenosine analog (IB-MECA) inhibits anchorage-dependent cell growth of various human breast cancer cell lines

A3 adenosine receptor agonists have been reported to influence cell death and survival. Here we report the effects of an A3 adenosine receptor agonist, IB-MECA, on the cell growth of human breast cancer cell lines, MCF-7 (estrogen receptor positive) and MDA-MB468 (estrogen receptor negative). Therefore, this study was aimed to investigate the expression and possible action of A3 receptor in the human breast cancer cell lines. IB-MECA, at 1-100 microM, resulted in a significant cell growth inhibition (P < 0.05) which reached the maximum at 48 h, in the cell lines. In both cell lines, agonist-induced effects were antagonized by pretreatment with a selective A3 adenosine receptor antagonist, MRS1220. Using RT-PCR method, further confirmation was provided by the presence of mRNA of A3 receptor in the cells. In addition, IB-MECA was able to inhibit forskolin-stimulated cAMP levels, which indicate the functional form of A3 receptor on the cell surface of these breast cancer cell lines. These results suggest that the inhibitory effect of IB-MECA on the growth of human breast cancer cell lines is mediated through activation of A3 adenosine receptor.     

In vivo growth kinetics of P388 and L1210 leukemias

The P388 lymphocytic leukemia and the L1210 lymphoid leukemia are used as test systems for putative cytotoxic drugs. These leukemias are also used to investigate the perturbation of cell cycle progression of various chemical compounds in more detail. There is little information on the normal growth kinetics in vivo of these leukemias. In the present report we therefore present the results from growth kinetic studies of P388 and L1210 leukemic cells growing in ascites form in mice. We used 3H-TdR autoradiography, DNA flow cytometry and the stathmokinetic method. During exponential growth both leukemias showed a growth fraction of unity. Whereas no significant cell loss was observed during the early growth phase of P388 cells, cell loss was indicated by a discrepancy between potential and actual doubling times during exponential growth of L1210 cells. During the phase of growth retardation, the proportion of G1 and G2 cells increased at the expence of a reduced S phase fraction in the P388 leukemia, whereas only small changes in cell cycle distributions were seen with time after inoculation of L1210 cells. An increasing discrepancy in the reduction of the S phase fraction and the 3H-TdRLI was seen in the P388 cells with time after inoculation. Thus, a majority of P388 cells with S phase DNA content were unlabelled during the late phase of growth restriction, indicating resting cells in S phase. A good correlation was found between the 3H-TdR LI and S phase fraction throughout the life history of L1210 cells, revealing considerable differences in in vivo growth kinetics between the two leukemias. Such differences should be considered when evaluating test results.     

Diethylglyoxal bis(guanylhydrazone), a potent inhibitor of mammalian S-adenosylmethionine decarboxylase. Effects on cell proliferation and polyamine metabolism in L1210 leukemia cells

The polyamines are cell constituents essential for growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in the polyamine biosynthetic pathway. Methylglyoxal bis(guanylhydrazone) (MGBG) is an anti-leukemic agent with a strong inhibitory effect against AdoMetDC. However, the lack of specificity limits the usefulness of MGBG. In the present report we have used an analog of MGBG, diethylglyoxal bis(guanylhydrazone) (DEGBG), with a much greater specificity and potency against AdoMetDC, to investigate the effects of AdoMetDC inhibition on cell proliferation and polyamine metabolism in mouse L1210 leukemia cells. DEGBG was shown to effectively inhibit AdoMetDC activity in exponentially growing L1210 cells. The inhibition of AdoMetDC was reflected in a marked decrease in the cellular concentrations of spermidine and spermine. The concentration of putrescine, on the other hand, was greatly increased. Treatment with DEGBG resulted in a compensatory increase in the synthesis of AdoMetDC demonstrating an efficient feedback control. Cells seeded in the presece of DEGBG ceased to grow after a lag period of 1–2 days, indicating that the cells contained an excess of polyamines which were sufficient for one or two cell cycles in the absence of polyamine synthesis. The present results indicate that analogs of MGBG, having a greater specificity against AdoMetDC, might be valuable for studies concerning polyamines and cell proliferation.     

Regulation of S-adenosylmethionine decarboxylase in L1210 leukemia cells. Studies using an irreversible inhibitor of the enzyme

A potent irreversible inhibitor of S-adenosylmethionine (AdoMet) decarboxylase, S-(5'-adenosyl)-methylthio-2-aminooxyethane (AdoMeSaoe), was used to study the regulatory control of this key enzyme in the polyamine biosynthetic pathway. Treatment of L1210 cells with the inhibitor completely eradicated the growth-induced rise in AdoMet decarboxylase activity, resulting in a marked decrease in cellular content of spermidine and spermine. The putrescine content, on the other hand, was greatly elevated. Although no detectable AdoMet decarboxylase activity was found in the L1210 cells after treatment with AdoMeSaoe, the cells contained 50-fold higher amounts of AdoMet decarboxylase protein, compared to untreated cells during exponential growth. Part of this increase was shown to be due to elevated synthesis of the enzyme. This stimulation appeared to be related to the decrease in cellular spermidine and spermine content, since addition of either one of the polyamines counteracted the rise in AdoMet decarboxylase synthesis. The synthesis rate was determined by immunoprecipitation of labeled enzyme after a short pulse with [35S]methionine. In addition to a protein that co-migrated with pure rat AdoMet decarboxylase (Mr approximately 32,000), the antibody precipitated a somewhat larger labeled protein (Mr approximately 37,000) that most likely represents the proenzyme form. Treatment of the L1210 cells with AdoMetSaoe also gave rise to a marked stabilization of the decarboxylase which contributed to the increase in its cellular protein content. Addition of spermidine did not significantly affect this stabilization, whereas the addition of spermine reduced the half-life of the enzyme to almost that of the control cells.     

Production of anti-self-H-2 antibodies by C3D2F1 mice hyperimmune to L cell/L1210 hybrids and L1210 leukemia cells

During the course of immunization of (C3H × DBA/2)F₁ mice (genotype H-2^k/b) with L cell (H-2^k/k)/L1210 leukemia cell (H-2^d/d) hybrids and L1210 leukemia cells, some of them produced a good titer of anti-self-H-2 (H-2^d) antibodies. Antigens recognized by this anti-self-H-2 antiserum were shown to be controlled by the H-2K-IA-IB-IJ-IE subregions of the H-2^d but not H-2^k nor H-2^b haplotypes of parental as well as F₁ origins and to have a tissue distribution identical to that of class 1 H-2 (H-2K/D) antigens.     

Galactosyltransferase activity and cell growth: uridine diphosphate (UDP)galactose inhibition of murine leukemic L1210 cells

UDPgalactose inhibits the growth of mouse leukemic L1210 cells. In calf serum supplemented Dulbecco's medium (CS-DMEM), 1.2 mM UDPgalactose (UDPgal) inhibited cell growth by 50% (IC50), and 5 mM UDPgalactose inhibited cell growth by 92%. Other nucleotide sugars as well as galactose, glucose, and galactose-1-phosphate had little or no effect on cell growth. Uridine nucleotides, which inhibit galactosyltransferase activity, protected L1210 cells from the growth inhibitory effect of UDPgalactose when both were added simultaneously to culture media. Unlike mouse 3T12 cells, in which no inhibition of cell growth was observed with heat-inactivated calf serum (HICS)-DMEM, 5 mM UDPgalactose inhibited L1210 cell growth in HICS-DMEM to the same degree as that observed in CS-DMEM. In contrast to 3T12 cells, L1210 cells secrete significant galactosyltransferase activity into the media. Complete inhibition of 3T12 cell growth by UDPgal was observed if HICS-DMEM medium was first conditioned by L1210 cells for 48 hours. No difference in cell growth or [3H]thymidine uptake was detected after 6 hours of exposure to UDPgalactose, but both were significantly decreased at 24 and 48 hours. Flow cytometric analysis of UDPgalactose effects on L1210 cells revealed no differences in the distribution of cells in G1, S, or G2-M of the cell cycle after 6 hours of incubation, but after 16 hours of UDPgalactose treatment, L1210 cells were arrested in early S phase. These cells were completely viable and morphologically similar to control L1210 cells. Normal growth was resumed when UDPgal was removed. The data suggest that UDPgalactose inhibition of cell growth requires extracellular galactosyltransferase activity and that the effect is mediated via the cell membrane.     

Alternol inhibits proliferation and induces apoptosis in mouse lymphocyte leukemia (L1210) cells

Germline mutations of the serine/threonine kinase LKB1 (also known as STK11) lead to Peutz–Jeghers syndrome (PJS) that is associated with increased incidence of malignant cancers. However, the tumor suppressor function of LKB1 has not been fully elucidated. We applied yeast two-hybrid screening and identified that a novel WD-repeat protein WDR6 was able to interact with LKB1. Immunofluorescence staining revealed that WDR6 was localized in cytoplasm, similar to the localization of LKB1. Expression of LKB1 was able to inhibit colony formation of Hela cells. Interestingly, coexpression of WDR6 with LKB1 enhanced the inhibitory effect of LKB1 on Hela cell proliferation. Consistently, WDR6 was able to synergize with LKB1 in cell cycle G1 arrest in Hela cells. Coexpression of WDR6 and LKB1 was able to induce a cyclin-dependent kinase (CDK) inhibitor p27^Kip1. Furthermore, the stimulatory effect of LKB1 on p27^Kip1 promoter activity was significantly elevated by coexpression with WDR6. Collectively, these results provided initial evidence that WDR6 is implicated in the cell growth inhibitory pathway of LKB1 via regulation of p27^Kip1.     

Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells

N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24 h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.     

Suppressive effect of adrenalectomy on growth of L1210 leukemic cells in ascites

This study was designed to evaluate the effect of adrenalectomy on growth of L1210 leukemic cells in ascites of BDF1 mice. Varying doses of 1.5 x 10(4), 5.0 x 10(5), and 1.5 x 10(6) viable tumour cells were inoculated intraperitoneally into groups of either adrenalectomized or sham-operated mice. At days 4 to 7 after the inoculation, adrenalectomized mice inoculated with 1.5 x 10(4) or 5.0 x 10(5) tumour cells had a smaller number of tumour cells in ascites than sham-operated controls. However, after inoculation of 1.5 x 10(6) cells, no significant differences were found at days 2 to 4 between adrenalectomized and sham-operated mice. The growth retardation by adrenalectomy was not observed in adrenalectomized mice supplemented with 4 or 6 micrograms dexamethasone per day per mouse. It suggested that the ablation of glucocorticoids was at least partially responsible for the growth retardation observed in adrenalectomized mice. Cell kinetic analysis revealed that the difference in a potential doubling time could not explain these results. Tumour retention in the peritoneal cavity was measured using [125I]-iododeoxyuridine-labelled tumour cells as a tracer. At days 4 to 6 after inoculation of 5.0 x 10(5) labelled cells, radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham-operated mice. This ratio was almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of sham-operated ones. Consequently, growth retardation observed in adrenalectomized mice resulted from an increase in tumour cell migration and/or in tumour cell death, but not from an increase in doubling time.     

Epithelial tissue confinement inhibits cell growth and leads to volume-reducing divisions

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