Survival mechanisms induced by 12-O-tetradecanoylphorbol-13-acetate in normal human melanocytes include inhibition of apoptosis and increased Bcl-2 expression.

Phorbol esters, which activate protein kinase C, stimulate the growth of normal human melanocytes yet inhibit the growth of most melanoma cells. We investigated whether apoptosis mediates the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on melanocyte and melanoma cell growth. Few apoptotic cells were present when melanocytes were cultured with TPA. Upon removal of TPA, the number of apoptotic cells increased over 10 days. Addition of TPA did not induce apoptosis in a metastatic melanoma cell line, Demel, although it strongly inhibited its growth. Protection of normal melanocytes from apoptosis was associated with high levels of Bcl-2. Following withdrawal of TPA from melanocytes, the expression of Bcl-2 decreased steadily. Bax and Bcl-X(L) levels did not differ between melanoma cells or melanocytes and were unaffected by the addition of TPA. These results suggest that TPA plays an important role in stimulating the growth of melanocytes by promoting anti-apoptotic mechanisms associated with high levels of Bcl-2.

Homology between egg white sulfhydryl oxidase and quiescin Q6 defines a new class of flavin-linked sulfhydryl oxidases.

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. Reduced proteins are the preferred thiol substrates of this secreted enzyme. The egg white oxidase shows an average 64% identity (from randomly distributed peptides comprising more than 30% of the protein sequence) to a human protein, Quiescin Q6, involved in growth regulation. Q6 is strongly expressed when fibroblasts enter reversible quiescence (Coppock, D. L., Cina-Poppe, D., Gilleran, S. (1998) Genomics 54, 460-468). A peptide antibody against Q6 cross-reacts with both the egg white enzyme and a flavin-linked sulfhydryl oxidase isolated from bovine semen. Sequence analyses show that the egg white oxidase joins human Q6, bone-derived growth factor, GEC-3 from guinea pig, and homologs found in a range of multicellular organisms as a member of a new protein family. These proteins are formed from the fusion of thioredoxin and ERV motifs. In contrast, the flavin-linked sulfhydryl oxidase from Aspergillus niger is related to the pyridine nucleotide-dependent disulfide oxidoreductases, and shows no detectable sequence similarity to this newly recognized protein family.

Regulation of the cell cycle at the G2/M boundary in metastatic melanoma cells by 12-O-tetradecanoyl phorbol-13-acetate (TPA) by blocking p34cdc2 kinase activity.

12-O-Tetradecanoyl phorbol-13-acetate (TPA) inhibits the growth of most malignant melanoma cells but stimulates the growth of normal human melanocytes. We previously showed that addition of TPA inhibits the growth of the human metastatic melanoma cell line, Demel, by blocking cells at both the G1/S and G2/M cell cycle transitions (D. L. Coppock et al., 1992, Cell Growth Differ. 3, 485-494). To examine the G2/M transition, we developed a method to synchronize the cells in early S phase using Lovastatin and mevalonate, followed by treatment with hydroxyurea (HU). TPA (30 nM) was effective in blocking cells from entering mitosis and reentering G1 when added up to the end of G2. These cells arrested in G2. Examination of the levels of cyclins A and B1 demonstrated that the levels of these cyclins were not limiting for entrance into M. However, the addition of TPA blocked the increase in p34(cdc2)/cyclin B1 kinase activity. In cells treated with TPA, most p34(cdc2) was found in the slowly migrating forms on Western blots, which contained increased levels of phosphotyrosine. In addition, the level of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1), but not of p27(Kip1), was increased. We examined the expression of protein kinase C (PKC) isoforms in Demel cells using Western blots to understand which types were involved in the G2 arrest. Demel cells expressed the PKC alpha, betaI, betaII, delta, epsilon, iota/lambda, zeta, and mu isozymes. PKC eta and PKC theta were not detected. Addition of TPA did not completely down regulate any PKC isozymes over a 12-h period in these synchronized cells. PKC alpha, betaI, betaII, delta, and epsilon isozymes were translocated to the membrane fraction from the cytosolic fraction when treated with TPA. PKC delta appeared as a doublet and the addition of TPA shifted a majority to the slower migrating form. The level of PKC mu was constant; however, a slow mobility form was observed in TPA-treated cells. This reduced mobility was at least partially due to phosphorylation. Thus, the arrest of growth in G2 appears to be due to the inhibition of the p34(cdc2) kinase activity which is associated with the increased expression of p21(Cip1/Waf1) and increased phosphorylation on tyrosine of p34(cdc2). This arrest, in turn, is associated with a shift of PKC isozymes PKC alpha, PKC betaI, PKC betaII, PKC delta, PKC epsilon, and PKC mu to the membrane fraction which is induced by addition of TPA.

The quiescin Q6 gene (QSCN6) is a fusion of two ancient gene families: thioredoxin and ERV1.

Cell and tissue growth is a dynamic process determined by the fraction of cells in the proliferative cycle, the fraction of cells in quiescence, and the rate of cell death. Genes whose expression is induced at the beginning of the transition from the proliferative cell cycle to quiescence may play an important role in this process. We have identified a gene, Quiescin Q6 (QSCN6), whose expression is induced just as fibroblasts begin to leave the proliferative cycle and enter quiescence. QSCN6 is located on human chromosome 1q24, near the putative hereditary prostate cancer locus (HPC1). A triplet repeat (CTG)n encodes a putative signal sequence. The gene encodes a 582-amino-acid open reading frame that has domains that are members of two ancient gene families. These domains apparently underwent a gene fusion event during metazoan evolution to create QSCN6. QSCN6 is most closely related to three genes of unknown function from Caenorhabditis elegans as well as a gene from guinea pig. Analysis of this relationship showed nine Quiescin homology zones (QHZ). QHZ 0 is the putative signal sequence, QHZ 1 is homologous to a thioredoxin domain, and QHZ 2, 3, 4, and 8 are homologous only to themselves, while QHZ 5, 6, and 7 are homologous to the ERV1 gene of Saccharomyces cerevisiae. In both thioredoxin and ERV1 gene superfamilies, QSCN6 sequences appear to be on distinct branches of their respective phylogenetic trees, consistent with an ancient origin of the QSCN6 gene. We present a model of the origin of QSCN6 and discuss its potential role in growth regulation.

Inhibition of the melanoma cell cycle and regulation at the G1/S transition by 12-O-tetradecanoylphorbol-13-acetate (TPA) by modulation of CDK2 activity.

The growth of malignant melanoma cells is inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) while the growth of normal melanocytes is stimulated. We previously demonstrated that TPA inhibits the growth of Demel melanoma cells and leads to arrest at both at the G1/S and G2/M cell cycle transitions. To investigate the mechanism by which TPA arrests melanoma cell growth at the G1/S transition we have examined its effects on the levels of cyclins and cyclin dependent kinases (CDKs) and activation of CDK2 kinase activity. Addition of TPA in G1 blocked the increase in the level of p34cdc2 mRNA, but not of CDK2 mRNA. When TPA was added in G1, it inhibited the mobility shift of CDK2 reflecting a change in phosphorylation state. This corresponded to inhibition of the increase in CDK2 histone H1 kinase activity. There was little effect on the level of CDK4. Treatment with TPA during G1 caused a three to four fold increase in cyclin D1 mRNA expression, but blocked the increase in the expression of cyclin A and cyclin B mRNAs later in the cell cycle. TPA caused a small increase in levels of cyclin D1 and had little effect on cyclin E, suggesting these G1 cyclins were not limiting. Addition of TPA in G1 prevented an increase in cyclin A levels, suggesting cyclin A might play an important role in mediating the growth inhibition. Examination of the levels of the CDK inhibitors p21Cip1 and p27Kip1 showed that the level of these inhibitors was higher in G1 and dropped as cells entered S phase. In the presence of TPA this decrease did not occur. These results demonstrate that TPA blocks the G1/S transition in Demel melanoma cells in late G1 by mechanisms which regulate phosphorylation and activation of the CDK2 kinase. These mechanisms include preventing the decrease in p21Cip1 and p27Kip1 kinase inhibitors and limiting the amount of cyclin A.

Identification of a malignant counterpart of the monocyte-dendritic cell progenitor in an acute myeloid leukemia.

Myeloblasts derived from the peripheral blood of a patient with acute myelogenous leukemia (ORL47) were found to represent the malignant counterpart of the newly elucidated monocyte-dendritic cell colony-forming unit (mono-DC-CFU). The specific cytokine conditions require to achieve intermediate and terminal maturation of DCs and monocytes from these progenitors were defined. With tumor necrosis factor (TNF) + granulocyte-macrophage colony-stimulating factor (GM-CSF) + stem cell factor treatment numerous colony-like clusters developed. In contrast with normal DC development, further advancement of mono-DC-CFU and terminal DC maturation from the leukemic cells were dependent on the addition of interleukin-6. Functional and phenotypic analysis showed that the capacity to differentiate was maintained fully in the DC compartment, but only partially in the monocyte compartment, as judged by the lack of CD14 surface expression. Cells found at intermediate stages of DC development were potent stimulators of a mixed leukocyte reaction, a function usually attributed to mature DCs. As previously shown for normal DC development, antibodies to TNF alpha and GM-CSF blocked proliferative responses and DC growth. The importance of these observations in the classification of leukemias, normal DC development, and potential clinical strategies is discussed.

Preferential gene expression in quiescent human lung fibroblasts.

The exit from the proliferative cell cycle into a reversible quiescence (G0) is an active process that is not yet well understood at the molecular level. Investigation of G0-specific gene expression is an important step in studying the mechanism regulating the entrance to quiescence. Using the human embryo lung fibroblast (WI38) as a model system, we have isolated complementary DNA clones that are expressed at a higher level in quiescent cells than in logarithmically growing cells. We have identified complementary DNAs from eight genes including collagen alpha 1(VI), collagen alpha 1(III), decorin, complement C1r, collagen alpha 1(I), collagen alpha 2(I), and two novel genes, Q6 and Q10. We have named this class of quiescence-inducible genes quiescins. Expression of these genes was induced just as proliferation slowed, as indicated by the level of histone H2B mRNA, [3H]-thymidine incorporation, and cell number. The level of expression of the novel genes, Q6 and Q10, increased at the same time as the other genes. Q6 has two mRNAs of 3 and 4 kb, whereas Q10 mRNA is about 1.0 kb. The expression of the quiescins was not induced by blocking the cell cycle in S phase with aphidicolin or in G1 with lovastatin. However, the genes were highly induced by trypsinization or scraping of the cells during logarithmic growth. This induction was not blocked by inhibitors of RNA synthesis. The expression of decorin and Q6 was very low in SV40-transformed cells (VA13) either in logarithmic growth or at high density, whereas the gene Q10 was expressed more highly in VA13 than in WI38 cells. The finding that expression of some components of the extracellular matrix is induced as cells enter G0 suggests that they may have a role in both the induction and the maintenance of the quiescent state. The quiescins will serve as molecular markers for the investigation of mechanisms that regulate the onset of quiescence.

Grass hay and Acacia fruits: a local feeding system for improved calf performance in semi-arid Ethiopia.

A 90-day growth trial was designed to compare the performance of calves on 3 dry-season diets composed of local resources from the Borana pastoral system. The control group received the traditional diet of cut-and-carry, standing-brown grass while the other diets consisted of grass hay stored since the previous wet season with or without Acacia tortilis fruits as a protein supplement. All calves had access to water once every 3 days as traditional. The objective was to see whether modest changes in traditional feeding management could enhance nutrient intake and growth of calves under conditions of restricted water access. The hay had a higher nitrogen content and in vitro digestibility than the standing grass, and the Acacia fruits had higher nutrient concentrations than the hay (both at P < or = 0.05). Calves on hay plus Acacia fruits had higher nitrogen intakes than those on hay only, and those on hay only had higher nitrogen intakes than those on standing grass (both at P < or = 0.05). Calves on standing grass lost weight and condition, those on hay only maintained weight but lost condition, and those on hay plus Acacia fruits gained weight and maintained condition (all at P < or = 0.05). Calves consumed the most feed on day 2 of the watering cycle, regardless of treatment. Water intake increased 27% for animals on both hay diets compared to those on standing grass (P < or = 0.01). Feeding packages based on hay making and collection of browse legumes are appropriate options for extension to these semi-settled pastoralists.

Homogeneously staining region in anthracycline-resistant HL-60/AR cells not associated with MDR1 amplification.

Anthracycline-resistant HL-60/AR cells and their drug-sensitive HL-60/S counterparts were characterized by karyotypic analysis and examined for the overexpression of DNA and mRNA sequences coding for P-glycoprotein (Pgp). The HL-60/S cells were karyotypically stable over a 5-year period of study (1986-1991), except for an additional small Giemsa-positive band noted at 7q22 in cultures harvested in 1987, but not in 1986. This change did not affect drug sensitivity. The drug-resistant HL-60/AR cells examined in 1986, 1987, and 1991 demonstrated a very stable karyotype. The most striking feature was a large homogeneously staining region in the long arm of chromosome 7 (7q11.2), and translocation of the remainder of the long arm to another centromere. Other changes in the HL-60/AR cells included inversion in 9q, partial deletion of the short arm of chromosome 10p, addition of material to the p arm of der(16), loss of chromosome 22, and the appearance of a new marker chromosome. Both HL-60/S and the HL-60/AR cells were found not to amplify DNA or mRNA sequences coding for the Pgp. Thus, although the HL-60/AR cells possess the classical multidrug resistance phenotype and demonstrate a homogeneously staining region near the region of the MDR1 gene, their resistance is due to mechanisms other than those coded for by MDR1.

12-O-tetradecanoylphorbol-13-acetate induces transient cell cycle arrest in G1 and G2 in metastatic melanoma cells: inhibition of phosphorylation of p34cdc2.

The growth of Demel human metastatic melanoma cells was inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA) and other nonphorbol tumor promoters including palytoxin and okadaic acid. Using flow cytometry, we have demonstrated that the cells arrested growth in G1 and G2 phases of the cell cycle. Detailed analysis of the kinetics of the growth arrest in unsynchronized cells showed that (a) the growth arrest was transient and peaked 16-20 h following addition of TPA; (b) effects of TPA on cell growth began within 1-2 h after the addition; and (c) cells completed S phase and arrested in G2. In addition, TPA induced a pronounced morphological change, which peaked by 1 h and gradually subsided over 24 h. In populations of cells synchronized in G1 using lovastatin, (a) addition of TPA blocked the onset of DNA synthesis up to the end of G1; (b) the lag between addition of the drug and onset of DNA synthesis was less than 30 min; and (c) addition of TPA at the end of G1 prevented the increased phosphorylation of p34cdc2, as determined by immunoprecipitation. The experiments reported here show that TPA transiently blocked the proliferation of Demel melanoma cells at the G1-S border and in G2, thus preventing cells from progressing through the cell cycle. These experiments suggest that pathways involving protein kinase C interact with and rapidly alter the molecular pathways involving p34cdc2 which regulate the onset of DNA synthesis and the G2-M transition.

Physiological responses of plant populations to herbivory and their consequences for ecosystem nutrient flow.

We explored how responses of two populations variable in grazing tolerance provide feedbacks to nutrient supply by controlling carbon supply to soil heterotrophs. The study focused on differences in production and carbon and nitrogen allocation patterns between the two populations. The grazing-tolerant population, or on-colony population, is found on intensively grazed prairie dog colonies, and a grazing-intolerant population, the off-colony population, is found in uncolonized grasslands. Equations describing the production and allocation responses to defoliation for the two ecotypes described were incorporated into CENTURY, a nutrientcycling simulation model. Simulations showed an increase in plant production that paralleled increases in net nitrogen mineralization. Production was greater with grazing and was maintained at higher grazing intensities for the on-colony than the off-colony population. Differences between the populations provided important controls over nitrogen losses. Feedbacks between plant responses to grazing and nitrogen cycling accounted for increased nitrogen availability with grazing. These feedbacks were more important determinants of ecosystem function than were fertilization effects of urine and feces deposition. The simulation results suggest that ecosystem function may be sensitive to physiological differences in population responses to periodic disturbances like herbivory.

Replication of Xenopus erythrocyte nuclei in a homologous egg extract requires prior proteolytic treatment.

Reactivation and reinitiation of DNA replication in quiescent frog erythrocyte nuclei has been analyzed following incubation in extracts prepared from activated Xenopus eggs. Nuclear decondensation and DNA synthesis only occurred if nuclei were pretreated with low doses of trypsin. This protease treatment did not digest histones, but did degrade several nonhistone proteins. Activated erythrocyte nuclei swell and begin DNA synthesis by 30 min after being mixed with the egg extract. In some extracts virtually complete genome replication was achieved in all nuclei after 2-3 hr. Addition of several protease inhibitors during sperm nuclear isolation significantly reduced the template efficiency of these preparations. We concluded that proteolytic alteration of nonhistone nuclear structural proteins may be a general mechanism which permits quiescent nuclei to reenter the replication cycle. Erythrocyte nuclei and egg extracts provide an excellent experimental system in which to investigate the processes of nuclear reactivation.

Control of thymidine kinase mRNA during the cell cycle.

To investigate the mechanism which controls the onset of DNA synthesis, we examined the regulation of thymidine kinase (TK) and its mRNA in the cell cycle. TK activity provides a useful marker for the onset of the S phase in mammalian cells. The present analysis of regulation of TK mRNA in BALB/c 3T3 cells showed that (i) the increase in TK activity depended on the availability of TK mRNA, (ii) the level of TK mRNA between G0 and S increased more than 20-fold, (iii) the rate of run-on TK transcription increased at most 2- to 4-fold between the G0 and S phases, (iv) the half-life of TK mRNA was greater than 8 to 12 h in the S and M phases and decreased as cells entered quiescence, (v) the TK mRNA increase was fully blocked by inhibition of protein synthesis by only 60%, (vi) this inhibition was completely effective for up to about 10 h following serum addition and progressively much less effective when the drugs were added later. These results suggest that the appearance of TK mRNA at the beginning of the S phase in serum-stimulated 3T3 cells is controlled not only by the rate of gene transcription but importantly also by the decreased rate of mRNA degradation. Similar mechanisms may be involved in regulation of the onset of DNA synthesis and the increase in TK mRNA since both are controlled in a manner consistent with a requirement for a labile protein.

Energy extraction and use in a nomadic pastoral ecosystem.

An analysis of annual energy flows in an arid tropical ecosystem inhabited by nomadic pastoralists provides insight into a subsistence life-style that has persisted in droughted environments for hundreds to thousands of years. Although a large fraction of the total energy consumed by the Ngisonyoka of Kenya followed a single pathway from plant to animal to human, they also harvested solar energy from a relatively diverse assemblage of energy flow channels. Energy utilization and conversion efficiencies were generally low, as the system is maintenance-rather than production-oriented. Energy flow to maintenance must be relatively high to support biotic responses that enable tolerance of abiotic variability and to stabilize energy flow under the stress of severe droughts. Energy utilization by the Ngisonyoka is therefore consistent with ecological patterns that promote rather than diminish ecological stability under stress.

Regulation of thymidine kinase activity in the cell cycle by a labile protein.

Previous studies have shown that the onset of DNA synthesis in Balb/c 3T3 cells appears to be regulated by a labile protein. We have found that induction of thymidine kinase (TK) activity, after quiescent cells are stimulated by the addition of serum, is similarly regulated by a labile protein. Eight hours after serum stimulation, a 6-h pulse of cycloheximide (CHM) caused an excess delay of 2 h in TK induction. A similar delay also was found in the induction of thymidylate synthase (TS). In contrast, the benzo(a)pyrene transformed 3T3 cell line, BP-A31, which had previously been shown to have no excess delay for the onset of DNA synthesis also had no excess delay for the induction of TK activity after a pulse of CHM. The induction of TK was inhibited by actinomycin D and dichlororibofuranosylbenzimidizole (DRB) suggesting a requirement for new RNA synthesis. It did not appear to depend on DNA synthesis as it was not blocked by aphidicolin. In conclusion, the induction of TK activity appears to be regulated by the same labile cellular signal as the onset of DNA synthesis, and to depend on an increase in the level of TK mRNA in late G1 or early S phase.

Growth control variant cell line having increased serum requirement and decreased response to platelet-derived growth factor: reversion by 5-azacytidine.

Variants of the mouse embryo fibroblast X melanoma hybrid clone 100A have been isolated by a procedure that selects against cells that are able to grow in medium containing low concentrations of serum plus insulin. Three variant clones derived from this selection were found to have a much higher serum requirement than the parental clone 100A cells, as evidenced by a very low rate of DNA synthesis and growth in medium containing low concentrations of serum. Two of the variants had approximately double the number of chromosomes as the parental cell line, while one had approximately the same number of chromosomes as the parental cells. One of the variants was very strongly reverted by 5-azacytidine but not by ethyl methanesulfonate, suggesting that it reverted by a nonmutational mechanism such as a stable change in DNA methylation. Analysis of the growth requirements in hormone-supplemented serum-free media of the 100A parent, the INS 471 variant, and revertants of the variant indicated that the variant had a specific deficiency in its growth response to platelet-derived growth factor (PDGF). PDGF dose-response curves obtained with the variant cells were shifted approximately an order of magnitude toward higher PDGF concentrations relative to PDGF dose-response curves obtained with the parental 100A cells. This quantitative increase in PDGF requirement of the INS 471 variant appears to explain the increased serum requirement of this variant. Equilibrium binding experiments performed with 125I-PDGF suggest that the variant does not have a decreased number of PDGF receptors.