Retinoic acid-promoted expansion of total cellular ATP pools in 3T3 cells can mediate its stimulatory and growth inhibitory effects

Exposure of Swiss 3T3 cells to micromolar quantities of β-all-trans-retinoic acid (RA) results in either inhibition of growth or stimulation of cellular responsiveness to mitogens, depending on the length of treatment. Inhibition of growth is produced by treatment of the cells with RA for at least 48 hours. The total cellular pools of adenosine 5′-triphosphate (ATP) are markedly increased after 48-hour RA treatment and dose dependence studies show a correlation between the expanded ATP pools and the inhibitory effects. The expansion of total cellular ATP pools by retinoic acid occurs throughout the cell cycle and parallels the cell cycle-dependent fluctuations in total cellular ATP pools of untreated cells. Studies of [³H]thymidine incorporation and labeling indices in intact cells and [³H]dTTP incorporation and labeling indices in isolated nuclei of RA-treated and control cultures suggest that cellular acid-soluble nucleotide pools mediate the inhibition of DNA replication in the 48-hour-RA-treated cells. The stimulatory activity of RA for mitogenic responsiveness is demonstrated by treatment of G₀/G₁-arrested 3T3 cells with micromolar levels of RA for a maximum of 18 hours resulting in the potentiation of phorbol myristate acetate (PMA)-stimulated transition into S phase of the cell cycle. Marked increases in total cellular ATP and UTP pools are produced by 18-hour treatment of G₀/G₁-arrested cells with RA, before their exposure to PMA.     

Amino acid pools in mitotic and interphase HeLa cells

Mitotic HeLa cells collected by shake-off synchronizing procedures were found to have elevated amino acid influx rates into the acid-extractable pool. Combined use of colchicine or chilling before testing abolished the increased uptake; high external concentrations of Mg²⁺ further enhanced it. Telophase and subsequent interphase populations showed a lower uptake rate which remained constant throughout most of the next cycle.     

Retinoic acid alters the metabolic 3H-labelling of glycosphingolipids

Retinoic acid increases the incorporation of radioactivity from a mixture of [3H]-galactose and [3H]-glucosamine into glycosphingolipids of serum-starved quiescent human foreskin fibroblasts with a preferential labelling of ceramide mono- and dihexoside as compared to ceramide tri- and tetrahexoside. Under the conditions used, no similar change in the specific labelling of glycoprotein is observed. Alteration in [3H]-precursor uptake into glycolipids comparable to that seen under the influence of retinoic acid does not occur in the presence of phorbolester, colchicine, butyrate or after infection with cytomegalovirus.     

Aging but not dietary restriction alters the activation-induced apoptosis in rat T cells

The aim of this study was to determine if aging or dietary restriction (DR) alters activation-induced cell death, which is known to regulate cell proliferation and eliminate the high number of activated cells during an immune response. Splenic T cells were isolated from young (4-6 months) and old (25-26 months) Fischer 344 rats that had free access to food, ad libitum (AL), and from dietary-restricted (DR) old (25-26 months) rats that beginning at 6 weeks of age were fed 60% (40% food-restricted) of the diet consume by the AL rats. T cells were incubated with anti-CD3 antibody, or staphylococcal enterotoxin B (primary stimulus) for 72-96 h, followed by restimulation with anti-CD3 (secondary stimulus) for 72 h. Activation-induced apoptosis was assessed by DNA fragmentation and the expression of Fas/CD95 receptor and Fas ligand (Fas-L) was measured by flow cytometry. We found that the amount of DNA fragmentation was significantly (P<0.05) higher in the stimulated and restimulated T cells from AL old rats and DR old rats compared to young rats. The increase in DNA fragmentation with age was paralleled by an increase in the proportion of the cells expressing Fas and Fas-L. However, DR had no significant effect on the age-related increase in DNA fragmentation or the expression of Fas or Fas-L. We also measured the levels of Bcl-2 and Bax protein and found that the level of Bcl-2 decreased and Bax increased with age and that DR had no effect on the age-related changes in the level of Bcl-2 or Bax protein. These results demonstrate that aging but not DR alters activation-induced apoptosis in rat T cells.     

Cell cycle-dependent subcellular distribution of ClC-3 in HeLa cells

Chloride channel-3 (ClC-3) is suggested to be a component and/or a regulator of the volume-activated Cl(-) channel in the plasma membrane. However, ClC-3 is predominantly located inside cells and the role of intracellular ClC-3 in tumor growth is unknown. In this study, we found that the subcellular distribution of endogenous ClC-3 varied in a cell cycle-dependent manner in HeLa cells. During interphase, ClC-3 was distributed throughout the cell and it accumulated at various positions in different stages. In early G1, ClC-3 was mainly located in the nucleus. In middle G1, ClC-3 gathered around the nuclear periphery as a ring. In late G1, ClC-3 moved back into the nucleus, where it remained throughout S phase. In G2, ClC-3 was concentrated in the cytoplasm. When cells progressed from G2 to the prophase of mitosis, ClC-3 from the cytoplasm translocated into the nucleus. During metaphase and anaphase, ClC-3 was distributed throughout the cell except for around the chromosomes and was aggregated at the spindle poles and in between two chromosomes, respectively. ClC-3 was then again concentrated in the nucleus upon the progression from telophase to cytokinesis. These results reveal a cell cycle-dependent change of the subcellular distribution of ClC-3 and strongly suggest that ClC-3 has nucleocytoplasmic shuttling dynamics that may play key regulatory roles during different stages of the cell cycle in tumor cells.     

Expression of Nox1 in 3T3 cells increases cellular acid production but not proton conductance

The role of the NADPH oxidase homolog 1 (Nox1) in plasma membrane H(+) conductance and cellular H(+) production was investigated in 3T3 cells stably expressing Nox1 (Nox1 3T3) compared to vector-expressing control cells (mock 3T3). In whole cell patch clamp experiments both Nox1 and mock 3T3 expressed a similar H(+) conductance (Nox1 3T3, 13.2+/-8.6 pS/pF; mock 3T3, 16.6+/-13.4 pS/pF) with a number of similar characteristics (e.g., current-voltage relations, current activation kinetics, Zn(2+)-sensitivity). When the intracellular pH of cells was alkalinized with NH(4)Cl, rates of intracellular acidification were significantly higher in Nox1 3T3 compared to mock 3T3. Nox1 3T3 showed a time course of acidification that followed a double-exponential function with a fast and a slow component of, on average, tau=165 s and 1780 s, whereas mock 3T3 showed only a single slow tau of 1560 s. Expression of Nox1 also caused cells to acidify the extracellular medium at higher rates than control cells; Nox1 3T3 released 96+/-19 fmol h(-1)cell(-1) of acid equivalents compared to 19+/-12 fmol h(-1)cell(-1) in mock 3T3. These data show that expression of Nox1 results in a mechanism that has the capacity to rapidly acidify the cytosol and generate significant amounts of acid. No significant effect of Nox1 expression on the plasma membrane H(+) conductance was found.     

Conditional expression of a truncated fragment of nonmuscle myosin II-A alters cell shape but not cytokinesis in HeLa cells

A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1-591, delta N592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and delta N592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and delta N592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed delta N592 did not bind to actin, suggesting that delta N592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of delta N592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of delta N592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not delta N592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.     

Retinoic acid inhibits phospholipid turnover and protein kinase C activity in RA-sensitive but not in RA-resistant cells

Treatment with 10(-5) M retinoic acid causes loss of anchorage-independent growth in src-transformed RR1022 cells but not in ras-transformed KNRK cells. In an effort to elucidate the mechanisms underlying this difference, we investigated the effect of RA on phospholipid turnover and PKC activity in these two cell lines. 10(-5) M RA treatment caused a drastic inhibition of 32P incorporation into PI and PA and a large increase in 32P incorporation into PC in RR1022 cells. Similar treatment of KNRK cells yielded no change in PC or PA labelling and a much smaller decrease in PI labelling. Furthermore, 10(-5) M RA treatment causes a large decrease in PKC activity in RR1022 cells (35% of control) but only a small decrease in KNRK cells (78% of control). We suggest that these effects are part of an altered signal transduction pathway which mediates the differential effects of RA on anchorage-independent growth in these two cell lines.     

Cytoplasmic and nuclear pyrimidine ribonucleotide pools in HeLa cells

A comparison of the rate of approach to equilibrium U:C labeling for two species of RNA whose base composition is known has been described previously as a method to determine the similarity or difference in the pyrimidine ribonucleotide pools from which the RNA species are synthesized. The procedure is now utilized to examine the pools used for the synthesis of HeLa cell nuclear RNA, mitochondrial RNA and poliovirus RNA. The results indicate that poliovirus RNA molecules derive from a pyrimidine precursor pool indistinguishable from that used for the synthesis of both heterogeneous nuclear RNA and 45 S ribosomal RNA in the nucleus, but that a separate and distinct pool is used for the synthesis of mitochondrial RNA. In addition, a comparison of the radioactivity ultimately found in UMP and CMP in a single species of RNA labeled in the presence or absence of actinomycin D shows that the conversion of uridine to cytidine nucleotides is significantly slowed in the presence of the drug. Direct measurement of the ratios of radioactivity in these RNA precursors in the acid-soluble pool confirm this observation.     

Retinoic acid induces transglutaminase activity but inhibits cornification of cultured epidermal cells

In cultured mouse epidermal basal cells, retinoic acid is a potent inducer of transglutaminase, the enzyme responsible for isodipeptide bond formation in protein cross-linking in the production of the cornified membrane during terminal differentiation. Paradoxically retinoic acid also inhibits the formation of the cross-linked envelope and greatly reduces the level of dipeptide bond formation in epidermal cells induced to differentiate by calcium. These results suggest a novel mechanism by which retinoids can modify transglutaminase activity and epidermal differentiation.     

Separate pyrimidine-nucleotide pools for messenger-RNA and ribosomal-RNA synthesis in HeLa S3 cells.

Kinetic analyses of mRNA and 28-S RNA labeling [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleosidase attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA. The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 muM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.     

Phosphorylation but not transport of sugars is enhanced in virus-transformed mouse 3T3 cells.

The transport and phosphorylation of 2-deoxy-D-glucose are separate and sequential events in both normal and virus-transformed 3T3 cells. The apparent enhancement of 2-dOG uptake by 3T3 cells accompanying virus transformation is not due to an effect on the transport process but to enhanced phosphorylation by intracellular kinases. Phosphorylation of 3-O-methyl-D-glucose does not occur in these cells. Both the rate and extent of transport of this glucose analog is the same in normal cells, SV40 virus-transformed cells and sarcoma virus-transformed cells. The appropriateness of using 3-O-MeG for studies of the glucose transport system of animal cells is examined and discussed.     

Heme deficiency causes apoptosis but does not increase ROS generation in HeLa cells

Mitochondria provide cellular energy supply via respiration and are the major sites for the generation of reactive oxygen species (ROS). Mitochondria also play a fundamental role in apoptosis. Heme is a key factor in mitochondrial function. Defective heme synthesis or altered heme metabolism is associated with numerous diseases. Here we investigated the molecular mechanism by which heme promotes HeLa cell growth and survival. We found that heme deficiency-induced apoptosis involves the release of cytochrome c and the activation of caspase 3. However, heme deficiency-induced apoptosis appears to occur by a unique mechanism distinct from those known to mediate mitochondrial-dependent apoptosis. Specifically, our data show that heme deficiency causes apoptosis in a pathway that is independent of ROS generation and the collapse of mitochondrial membrane potential. These results provide insights into how defective heme synthesis or altered heme metabolism causes diseases and how heme may control cell growth and cell death.     

Localization of cap-binding protein in subcellular fractions of HeLa cells

The 26,000-M(r) cap-binding protein was analyzed by a cross-linking assay in cell fractions from uninfected and poliovirus-infected HeLa cells. Cap-binding protein was found in the postribosomal supernatant (S-200) and in the ribosomal salt wash. The cap-binding protein in the S-200 had a sedimentation coefficient of 5 to 7S and lacked the ability to restore translation in extracts of poliovirus-infected cells.     

Anandamide stimulates phospholipase D activity in PC12 cells but not in NIH 3T3 fibroblasts

The endogenous cannabinoid arachidonoylethanolamide was previously reported to have no effects on the phospholipase D activity in Chinese hamster ovary cells expressing the human brain-specific cannabinoid receptor, while in mouse peritoneal cells, delta9-tetrahydrocannabinol stimulated this enzyme. In this work, arachidonoylethanolamide (0.1-1 microM) was found to stimulate the phospholipase D-mediated phospholipid hydrolysis in rat adrenal pheochromocytoma PC12 cells, but not in mouse NIH 3T3 fibroblasts. The phospholipase D-activating effects of arachidonoylethanolamide were comparable to those elicited by phorbol ester and nerve growth factor, while arachidonic acid (1 microM) had no effects. The results show that, depending on the cell type, arachidonoylethanolamide can be an activator of the phospholipase D system.     

Inhibition of post-replication repair of alkylated DNA by caffeine in Chinese hamster cells but not HeLa cells

Caffeine has been found to potentiate the lethal effects of sulphur mustard (SM) and N-methyl-N-nitrosourea (MNU) in a line of Chinese hamster cells but not in a line of HeLa cells. The sensitization of SM-treated cells by caffeine was S phase specific, and persisted for up to 24 h after alkylation of asynchronous cell cultures. The sensitization of MNU-treated cells, however, was not S phase specific but persisted for up to 50 h after the initial alkylation. Possible explanations for this difference between these two types of alkylating agent were discussed. Previously, evidence was presented which suggested that the alkylation-induced delay in the time of the peak rate of DNA synthesis in Chinese hamster cells was associated with the operation of post-DNA replication repair mechanism in these cells. Caffeine has now been found to reverse this alkylation-induced delay of DNA synthesis in both SM- and MNU-alkylated Chinese hamster cells. It is therefore proposed that caffeine sensitizes alkylated cells by inhibition of a post-replication DNA repair mechanism. No support was obtained for the alternative possibility that caffeine inhibits alkylation-induced excision repair of damaged DNA. The role of DNA repair in the production of the lethal mutagenic and cytological effects of alkylating agents is discussed.     

Geranylgeranylated, but not farnesylated, RhoB suppresses Ras transformation of NIH-3T3 cells

RhoB is a low molecular weight GTPase that is both farnesylated (RhoB-F) and geranylgeranylated (RhoB-GG) in cells. Based on data from rodent cell models, it has been suggested that RhoB displays differential effects on cell transformation, according to the nature of its prenylation. To test directly this hypothesis, we generated GTPase-deficient RhoB mutants that are exclusively either farnesylated or geranylgeranylated. We show that in Ras-transformed murine NIH-3T3 cells, RhoB-F enhances, whereas RhoB-GG and RhoB (F/GG) suppresses anchorage-dependent and -independent cell growth as well as tumor growth in nude mice. We then demonstrate that Ras constitutive activation of the tumor survival pathways Akt and NF-kappa B are blocked by RhoB-GG, but not by RhoB-F, providing further support for the opposing role of RhoB-F and RhoB-GG in Ras malignant transformation in NIH-3T3 cells. In addition, both RhoB (F/GG) and RhoB-GG induce apoptosis in Ras-transformed NIH-3T3 cells whereas RhoB-F has no effect. Our data demonstrate that RhoB-F and RhoB-GG which differ only by a 5-carbon isoprene behave differently in rodent cells highlighting the important role of prenyl groups in protein function and emphasize the potency of RhoB to regulate negatively the oncogenic signal.     

Retinoic acid down-regulates VPAC(1) receptors and TGF-beta 3 but up-regulates TGF-beta 2 in lung cancer cells

The effects of retinoic acid (RA) on lung cancer cells were investigated. Both all-trans (t-RA) and 13-cis RA (c-RA) decreased specific ¹²⁵I-VIP binding to NCI-H1299 cells in a time- and concentration-dependent manner. After 20 hr, 30 μM t-RA decreased specific ¹²⁵I-VIP binding by 60%. By Scatchard analysis, the density of VIP binding sites but not the affinity was reduced by 42%. NCI-H1299 VPAC₁ receptor mRNA was reduced by 48%. VIP caused a 3-fold elevation in the NCI-H1299 cAMP, and the increase in cAMP caused by VIP was reduced by 38% if the NCI-H1299 cells were treated with t-RA. Using the MTT assay, 3 μM t-RA and 3 μM c-RA inhibited NCI-H1299 proliferation by 60 and 23% respectively. Also, transforming growth factor (TGF)-β2 increased after treatment of NCI-H1299 cells with t-RA whereas TGF-β1 mRNA was unaffected and TGF-β3 mRNA was decreased. These results suggest that RA may inhibit lung cancer growth by down-regulating VPAC₁ receptor and TGF-β3 mRNA but up-regulating TGF-β2 mRNA.