Non-synchronized Cells Research Articles

Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule.

We have previously shown that a 20 amino acid peptide derived from the third ankyrin-like repeat of the p16CDKN2/INK4a (p16) tumour suppressor protein (residues 84-103 of the human p16 protein) can bind to cdk4 and cdk6 and inhibit cdk4-cyclin D1 kinase activity in vitro as well as block cell cycle progression through G1. Substitution of two valine residues corresponding to amino acids 95 and 96 (V95A and V96A) of the p16 peptide reduces the binding to cdk4 and cdk6 and increases its IC0.5 for kinase inhibition approximately threefold when linked to the Antennapedia homeodomain carrier sequence. The same mutations increase the IC0.5 approximately fivefold in the p16 protein. Substitution of aspartic acid 92 by alanine instead increases the binding of the peptide to cdk4 and cdk6 and the kinase inhibitory activity. The p16 peptide blocks S-phase entry in non-synchronized human HaCaT cells by approximately 90% at a 24 microM concentration. The V95A and V96A double substitution minimizes the cell cycle inhibitory capacity of the peptide whereas the D92A substitution increases its capacity to block cell cycle progression. A deletion series of the p16 derived peptide shows that a 10 residue peptide still retains cdk4-cyclin D1 kinase and cell cycle inhibitory activity. The p16 peptide inhibited S-phase entry in five cell lines tested, varying between 47-75%, but had only a limited (11%) inhibitory effect in the pRb negative Saos-2 cells at a concentration of 24 microM. Like the full length p16 protein, the p16 peptide does not inhibit cyclin E dependent cdk2 kinase activity in vitro. These data suggest that acute inhibition of CDK-cyclin D activity by a peptide derived from the INK4 family will stop cells in late G1 in a pRb dependent fashion.

Reconstruction of the Centrosome Cycle from Cryoelectron Micrographs

The absence of detailedin vitrostudies leaves the molecular events involved in the centrosome cycle poorly characterized. Most earlier studies have employed electron microscopy of thin or thick sections of cells. Here we have analyzed the structure of centrosomes isolated from nonsynchronized human lymphoblastic KE37 cells using cryoelectron microscopy of vitrified specimens. The centrosomes were classified into five categories depending on the number of centrioles (one or two), the respective orientation of the two centrioles in a pair (orthogonal or disoriented), and the presence or absence of appendages at the distal extremity of the centrioles (referred to as mature and immature, respectively). A detailed analysis of the centriole dimensions in these categories allowed us to reconstruct the centrosome cycle in KE37 cells. Our results suggest that centriole assembly is completed only when the mother centriole of an immature orthogonal pair separates from its daughter in preparation to centrosome duplication. Our study shows that anin vitroapproach based on cryoelectron microscopy of vitrified specimens can be used to obtain detailed structural information on the centrosome cycle.

Cell cycle dependence of radiation-induced homologous recombination in cultured monkey cells

A lacZ transgene recombination system that reports homologous recombination events involving duplicated lacZ segments was used to study recombination in monkey cells exposed to ionizing radiation at different points in the cell cycle. With this system, recombination events can be detected in single cells by histochemical staining soon after exposure of cells to DNA-damaging treatment. Ionizing radiation rapidly induced recombination 5–10-fold in cells that were at the mitosis stage of the cell cycle. Irradiation either of cells at other points in the cell cycle or of nonsynchronized cells had less of an effect on recombination between lacZ segments.

Deletion of complex gangliosides of human glioma cells during mitotic cell division.

Glycolipid compositions of the human glioma cell line T98G were studied during each phase of the cell cycle to see if those cell surface molecules are concerned with cell proliferation. In vitro cultured non-synchronized T98G cells are composed of ceramidemonohexoside (CMH), ceramidedihexoside (CDH), ceramidetrihexoside (CTH) and neolactotetraosylceramide (nLc4Cer) as neutral glycolipids, and of sulfatide (CS), gangliosides GM3, GM2, GD1a and several other gangliosides as acidic ones. While total glycolipid content per cellular weight was shown to be increased during the M phase, deletion of complex gangliosides particularly b-series gangliosides was recognized (p < 0.05). The glycolipid profile in other phases was fairly consistent, and there was no glycolipid molecule specific to a certain phase of the cell cycle. Relative enhancement of simple gangliosides with a decrease of complex ones during mitotic division may imply the functional involvement of complex gangliosides in cell-cell or cell-matrix attachment, which may have to be abandoned during the process of detachment from the matrix or cellular cleavage.

Inhibition of Pgp activity and cell cycle-dependent chemosensitivity to doxorubicin in the multidrug-resistant LoVo human colon cancer cell line.

To determine whether the cell cycle affects multidrug resistance (MDR) and its reversal, doxorubicin (DOX) cytotoxicity and the effect of inhibition of P-glycoprotein (Pgp) activity by verapamil (VER) were investigated in MDR LoVo cell lines (LoVo-R) in different phases of the cell cycle. Synchronised cells were obtained by exposing cells for 24 h to non-toxic concentrations (40 nmol/l) of methotrexate (MTX), which induced a reversible blockade in the S phase. DOX cytotoxicity was higher if cells were exposed to DOX shortly after the pretreatment with MTX, when most cells were in the S phase of the cell cycle. At that time, the DOX concentration inhibiting cell growth by 50% (IC10) was decreased by approximately 4-fold compared to non-synchronised cycling cells. DOX cytotoxicity remained high during the transition from the S to the G2M phase, but was reduced when the cells had shifted to the G2M phase. Inhibition of Pgp activity by VER (6 mumol/l) enhanced DOX uptake and resulted in an intracellular nuclear compartmentalisation of DOX in LoVo-R cells. These effects were not significantly different (P = NS) in the different phases of the cell cycle. However, similar increases in intracellular DOX uptake due to the inhibitory effect of VER on Pgp greatly potentiated DOX cytotoxicity in LoVo-R cells synchronised in the S or G2M phase compared with non-synchronised cycling cells. The ratio between DOX IC50 in the absence and presence of VER in LoVo-R cells synchronised in the S phase and in cycling cells was 11.1 and 4.1, respectively (P < 0.01). This greater potentiation could be explained by the increased chemosensitivity of the S- and G2M-phase cells to intracellular DOX concentration compared with the non-synchronised cells. Finally, the combination of synchronisation by MTX and of inhibition of Pgp activity by VER produced a considerable reduction in DOX IC50 (approximately 50-fold) in LoVo-R cells compared with the cells not treated with MTX and VER. In conclusion, this study demonstrates that, in LoVo-R cells, the effect of Pgp inhibition on DOX cytotoxicity is dependent on cell cycle phase. DOX cytotoxicity is maximal when inhibition of Pgp activity occurs during the S/G2M phases.

Ionic signals in T47D human breast cancer cells

Increasing evidence that ion channels play a key role in the modulation of cellular mitogenesis led us to investigate the membranes of T47D human breast cancer cells to identify the ion currents present. We report here the results of voltage-clamp studies in the whole-cell configuration on isolated, non-synchronized single cells obtained from a ductal breast carcinoma. In these studies we identified an outward rectifying potassium current and a chloride current. The potassium current activated at potentials more positive than −40 mV, reached an average value of 1.4 nA, and did not inactivate with time. This current was sensitive to block by extracellular tetraethylammonium chloride (TEA, IC 50 = 1 μM), was insensitive to charybdotoxin (CTX, IC 50 = 7.8 μM), and was not diminished by repetitive pulses separated by 1 s. Rapid voltage-dependent inactivation of the current was demonstrated by tail current analysis. The current appeared calcium-insensitive. Application of hyperpolarizing pulses did not elicit an inward potassium rectifier current. Treatment with tetrodotoxin did not reveal the presence of an inward sodium current. The potassium current was increased by the presence of aspartate in place of chloride and in the presence of the chloride channel blocker 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). We conclude that currents present in T47D breast cancer cells include a chloride current and a voltage-gated potassium outward rectifier. We suggest that the potassium current, either alone or in conjunction with potassium currents reported in different human breast cancer cell lines by others, may play a role in the modulation of the cell cycle.

Dictyostelium mucoroides cells exhibit cell cycle-dependent sorting behavior as observed in D. discoideum development.

Evidence has been obtained indicating that the cell's position in the cell cycle at the onset of starvation is a naturally occurring variable closely involved in the subsequent sorting and pattern formation during the development of Dictyostelium discoideum Ax2. It is of interest to know whether a similar phenomenon is also noticed in species other than D. discoideum and also without any treatment of cells for cell synchronization. For this, the sorting behavior of D. mucoroides-7 (Dm7) cells and its relation to the cell-cycle phase at the onset of starvation were analyzed, using non-synchronized Dm7 cells pulse-labeled with 5'-bromo-2-deoxyuridine (BrdU). The results demonstrate that Dm7 cells starved at the early G2 phase aggregate most rapidly, but are eventually sorted out to the posterior prespore zone of migrating slugs. In contrast, cells starved at the mid late G2 phase exhibited slower aggregation, but were sorted out to the anterior zone (tip), this being basically similar to the sorting behavior of D. discoideum cells. Measurements of cell numbers and nuclearity provided evidence that approximately 80% of cells progressed their cell-cycle after the formation of multicellular structures (mounds), probably coupling with prespore differentiation as in the case of D. discoideum. Thus, cell cycle-dependent sorting during Dictyostelium development is most likely to be a common phenomenon in different species.

Argyrophilic proteins in Dinoflagellates: An ultrastructural, cytochemical and immunocytochemical study

Dinoflagellate protists constitute an original eukaryotic phylum and have an ancestor in common with ciliates. They are important tools in studies of structure and function of the nucleus because they present a mixing of prokaryotic characteristics such as chromatin devoid of histones and nucleosomes, eukaryotic characteristics such as the presence of a nuclear membrane, nucleoli and AgNOR-like proteins and original characteristics of their own. Among them are the permanent compaction of the chromosomes, the presence of a nuclear envelope during the whole cell cycle, rare bases in their DNA, as well as an original mitosis. We have studied the distribution of the nuclear argyrophilic proteins (AgP) in three genera of Dinoflagellates ( Prorocentrum, Crypthecodinium and Amphidinium) by means of light microscopy (LM) and electron microscopy (EM), using cytochemical silver staining and immunocytochemical reactions following various preparation procedures. By means of the silver staining reaction, we determined by LM the distribution of nucleoli in the three non-synchronized cell populations and localized by EM the presence of AgP. These are always found in the nucleolar fibrillo-granular compartment (FG) and partly in the chromosomes and in the nucleolar UCh (unwound region of the nucleolar chromosome corresponding to the NOR); the chromosomes and the UCh are always stained in P micans, under special conditions in C cohnii but never in A carterae. To determine whether these nucleolar and chromosomal proteins are similar or different, we modified the conditions of the silver staining reaction by acidic, alkaline or enzymatic pretreatments and changes in the reaction's temperature. Our results suggested that these proteins belong to different groups. We have characterized one of these proteins using a mammalian anti-B23 Ab in P micans cells. Positive labeling was mostly detected in chromosomes and UCh and in a smaller amount in the nucleolar FG and G compartments, co-locating with end-products of the silver staining reaction. This suggests that: i) one among the dinoflagellate chromosomal AgP is analogous to the B23 mammalian protein; and ii) this B23-like protein is probably a DNA partner.

Specific expression of a gene encoding a novel calcium-binding protein, CAF-1, during transition of Dictyostelium cells from growth to differentiation.

The gene expressions involved in the transition from cell proliferation to differentiation were analyzed, using synchronized Dictyostelium discoideum Ax-2 cells and the differential plaque hybridization method. As one of the genes (cDNA) specifically expressed when Ax-2 cells were starved just before the putative shift (PS)-point (putative shift point; a switchover point from growth to differentiation in the cell cycle), calfumirin-1 (CAF-1) was cloned, which encoded a novel calcium-binding protein with E-F hand. Although CAF-1 mRNA was slightly expressed in vegetatively growing cells, the expression was markedly increased in response to starvation of cells just before the PS-point. Northern analysis using non-synchronized Ax-2 cells showed that the CAF-1 mRNA is predominantly expressed within a few hours of starvation. Such a starvation-induced early expression of the CAF-1 mRNA raised a possibility that CAF-1 might be one of Ca2+ -binding proteins involved in the phase-shift of cells from growth to differentiation.

Effects of 50 Hz magnetic fields on C-myc transcript levels in nonsynchronized and synchronized human cells.

The effects of 50 Hz electromagnetic fields (EMFs) on the expression of the c-myc oncogene, known to be involved in normal cell proliferation and possibly also in tumor processes, were investigated in nonsynchronized human lymphoid cells immortalized by Epstein-Barr virus. Viral injury to such cells makes them a good model for exploring the possible cancer-promoting effects of 50 Hz magnetic fields. Parallel experiments were conducted on human HL60 leukemic cells. Cells were exposed to sinusoidal 50 Hz EMFs at 10 microT or 1 mT for 20 min, 1 h, 24 h, or 72 h. Exposure was performed either immediately after refeeding or 1.5 h after refeeding. C-myc transcript values were assessed by Northern blot analysis and normalized to those of the noninducible gene GaPDH. No statistically significant difference between the c-myc transcript levels of control and exposed cells was found in lymphoid or leukemic cells under our experimental conditions, either after short exposures of 20 min and 1 h or after longer exposures of 24 and 72 h. Other experiments were carried out with pseudosynchronized cells in an attempt to establish whether cells were especially sensitive to 50 Hz magnetic field exposure in any particular phase of the cell cycle. Accordingly, cells were pseudosynchronized in G0/G1 by serum deprivation and exposed for 20 min to a 50 Hz magnetic field, at 10 microT for lymphoid cells and 1 mT for HL60 cells. No significant difference was observed between the c-myc transcript levels of control and exposed cells for either of the synchronized cell types. These results for synchronized cells correlated with those for nonsynchronized cells.

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells.

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.

Cell Cycle-Dependent Localization of Irnmunoreactive Basic Fibroblast Growth Factor to Cytoplasm and Nucleus of Isolated Ovine Fetal Growth Plate Chondrocytes

Basic fibroblast growth factor (basic FGF) is a potent mitogen for chondrocytes in vitro and is present in developing cartilage in vivo. Studies of intracellular basic FGF localization in other cell types have revealed a transient nuclear presence. We have examined ovine fetal growth plate chondrocytes for the presence of intracellular basic FGF by immunocytochemistry. Chondrocytes were isolated from the proximal tibial growth plate of lamb fetuses between 75 and 80 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. In non-synchronized cell cultures 58 +/- 6% of cells (mean +/- s.d., n = 3) demonstrated cytoplasmic staining for immunoreactive basic FGF. Of these cells, 18 +/- 3% also exhibited strong nuclear staining. Chondrocytes were growth-restricted and restarted into the cell cycle with 2% (vol/vol) fetal calf serum. The timing of S phase was followed by nuclear labelling of nuclei with [3H] thymidine followed by autoradiography, or by the incorporation of [3H] thymidine into trichloroacetic acid-precipitable DNA in parallel cultures. A cytoplasmic presence of immunoreactive basic FGF did not appear, following immunocytochemistry, until the second half of G1 with 97% of cells immunopositive 2 hr prior to S phase. Nuclear staining for basic FGF appeared 2 hr before peak nuclear labelling index, and 56% of cell nuclei were immunopositive. Following entry into S phase cytoplasmic and nuclear basic FGF immunostaining rapidly disappeared. When these experiments were repeated with or without the presence of anti-basic FGF antibody or heparin, the presence of the antibody significantly reduced peak [3H] thymidine incorporation into DNA during S phase while exposure to heparin increased this. However, the proportion of cells demonstrating cytoplasmic or nuclear staining for immunoreactive basic FGF, and the time of onset of staining, were unaltered. Incubation of cells with suramin blocked subsequent DNA synthesis and no intracellular basic FGF was visualized. Cell-conditioned culture medium, extracellular matrix and cytoplasm from synchronized cultures of chondrocytes were taken at time points throughout the cell cycle and assessed for basic FGF content by radioimmunoassay. Basic FGF was detectable in each compartment and steadily rose throughout the second half of G1 to reach maximum values around the S phase. The accumulation of basic FGF in medium, matrix and cytoplasm was blocked by the presence of cycloheximide.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet-Derived Growth Factor (PDGF) Accelerates Induction of Competence, and Heparin Does Not Inhibit PDGF-Induced Competence in Primary Cultured Smooth Muscle Cells of Rat Aorta

The time-dependent effect of platelet-derived growth factor (PDGF) on cell proliferation was investigated to clarify whether PDGF accelerates the rate of proliferation or its start in primary cultured smooth muscle cells (SMC) of rat aorta. In synchronized SMC at the Go phase, 1, 10 and 100 ng/ml PDGF started DNA synthesis at 24, 15–18 and 12 hr, respectively, after stimulation by 3% fetal bovine serum (FBS) or hypophysectomized rat plasma (deficient in insulin-like growth factor-I (IGF-I) ). Heparin (1, 10 or 100 μg/ml) decreased only the rate of DNA synthesis stimulated by PDGF in synchronized SMC. DNA synthesis in non-synchronized cells stimulated by PDGF with FBS was determined up to 10 days in culture. The stimulation with 1% FBS plus 30 ng/ml PDGF potentiated the DNA synthesis which was saturated with stimulation by 10% FBS alone, suggesting that prolonged treatment of PDGF transforms SMC. These results demonstrated that PDGF concentration-dependently accelerated the induction of competence independently of IGF-I, and heparin did not inhibit PDGF-induced competence but inhibited progression in primary cultured SMC of rat aorta.

Minute virus of mice non-structural protein NS-1 is necessary and sufficient for trans-activation of the viral P39 promoter.

The genome of the autonomous parvovirus minute virus of mice (MVM) is organized in two overlapping transcription units: the genes coding for the two non-structural proteins (NS-1 ad NS-2) are transcribed from a promoter (P04) located at map unit 4, whereas the promoter controlling the capsid protein genes (P39) lies at map unit 39. We studied the effect of viral proteins on the activity of the P39 promoter in vivo. By site-directed mutagenesis we constructed clones encoding only one of the two NS proteins. The activity of the P39 promoter was measured in HeLa or EL-4 cells transfected with these clones, either by an RNase protection assay or by following the expression of a reporter gene, CAT (which codes for chloramphenicol acetyltransferase), placed under the control of this promoter. We found that the P39 promoter of strain MVMi is activated in trans by a viral gene product, and evidence to suggest that NS-1 is the only viral gene product responsible for this trans-activation. We also determined that the mechanism of trans-activation is very rapid, since all species of viral mRNAs appear together in non-synchronized infected EL-4 cells within a 2 h interval.

Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5-bromo-2'-deoxyuridine and propidium iodide.

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5-bromo-2'-deoxyuridine (BrdUrd) and double-stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 micrograms/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti-BrdUrd and PI. PGE1-treated cells monitored at 3-hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti-BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd-negative "S phase" cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move "backwards" in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to over-estimation of S phase cells in kinetic studies because of contaminating mitotic cells.

Intracellular Carbon Partitioning in Chlamydomonas reinhardtii

Using enzymic and isotope techniques the intracellular partitioning of newly fixed carbon was studied in synchronized cells of Chlamydomonas reinhardtii. Starch and growth metabolism, i.e. the use of carbon in biosynthesis, were found to be the major sinks for photosynthetically fixed carbon in the alga. Sucrose does not accumulate in significant quantities. The amount of carbon partitioned either into starch or growth varies during the 12 hour light/12 hour dark cell cycle. Starch is accumulated at the beginning and at the end of the light period while a net breakdown is observed in the middle of the light period and in the dark. In contrast, nonsynchronized cells accumulate starch all the time in the light which suggests that carbon partitioning is controlled by the cell cycle. Labeled bicarbonate is incorporated into starch even at times when the total intracellular level of starch is decreasing. This indicates a turnover of the starch pool in the light with synthesis and degradation occurring simultaneously and at different rates.

The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the CDC25 start gene product.

In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature. In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion. Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides. These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.

The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the CDC25 start gene product.

In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature. In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion. Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides. These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.

Cyclic AMP levels and cellular kinetics during maturation of human promyelocytic leukemia cells.

Differentiation of human promyelocytic leukemia cells (HL-60) in response to several classes of inducing agents is characterized by the sequential appearance of granulocytic or monocytic markers. Compounds that increase intracellular cAMP in HL-60 cells induce a program of maturation in which cells demonstrate early functional phagocytic properties. Cyclic nucleotide metabolism was studied during monocytic and granulocytic differentiation of the HL-60 cell line. In synchronous and nonsynchronous cell cultures, cyclic AMP levels were raised 300-fold or 25-fold in response to N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphate or the combination of prostaglandin E2 and theophylline, respectively. No reproducible changes in intracellular adenosine 3':5'-cyclic monophosphate levels occurred in response to retinoic acid or dimethyl sulfoxide, suggesting that changes in adenosine 3':5'-cyclic monophosphate levels alone do not mediate cell maturation induced by these compounds. Agents that increased intracellular cyclic AMP in HL-60 cells slowed the progress through the S and G2-M phase of the cell cycle, whereas other agents such as dimethyl sulfoxide stimulated cells through this same period.