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PubMed2000

Opioid growth factor regulates the cell cycle of human neoplasias.

The native opioid growth factor (OGF), [Met5]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and migration, as well as tissue organization, during development, cancer, homeostatic cellular renewal, wound healing, and angiogenesis. OGF action is mediated by the OGF receptor (OGFr). To investigate the target of OGF as to cell proliferation, the effects of excess OGF, and a deprivation of OGF-OGFr interaction by an opioid antagonist, naltrexone (NTX), were examined in 3 human cancer cell lines: pancreatic (BxPC-3), colon (HT-29), and head and neck (CAL-27). OGF exposure decreased growth, DNA synthesis, and mitosis, and increased the doubling time from control levels. FACS analysis revealed a marked increase in cells in the G0/G1 phase and compensatory reduction in cells in S and G2/M phases. Consistent with this observation, the percentage of labeled mitosis (PLM) analysis showed a notable increase in the time of the G0/G1 phase. Receptor blockade with NTX increased the rate of growth, length of DNA synthesis and mitotic phases, and decreased doubling time from control values. FACS analysis indicated an increase in the proportion of cells in S and G2/M phases, and a decrease in the number of cells in the G0/G1 phase. PLM evaluation demonstrated a shortening of the length of the S and G2 phases in the 3 cell lines, and decreases in the M and G0/G1 phases in some cancers. These results indicate that OGF action is directed at the G0/G1 phase, but interruption of OGF-OGFr interfacing has widespread repercussions on the cell cycle. The data on blockade of OGF-OGFr during log phase growth suggest a requisite escorting of the growth peptide and its receptor through the cell cycle.

Adenocarcinoma↗
PubMed2005

Nonsteroidal anti-inflammatory drug effects on osteoblastic cell cycle, cytotoxicity, and cell death.

Previous studies indicated that nonsteroidal anti-inflammatory drugs (NSAIDs) suppress bone repair, growth, and remodeling in vivo. Our previous in vitro study demonstrated that indomethacin and ketorolac inhibited osteoblast proliferation. In this study, we further investigated the influences of 4 NSAIDs on cell cycle kinetics, cytotoxicity, and cell death pattern in osteoblast cultures from rat fetal calvaria. Our results showed that NSAIDs significantly arrested cell cycle at the G(0)/G(1) phase and induced cytotoxicity and cell death of osteoblasts. Apoptosis was more pronounced than necrosis caused by NSAIDs. Among these NSAIDs, piroxicam showed the least effect to produce osteoblastic dysfunction. Moreover, we found that the cytotoxic and apoptotic effects of NSAIDs on osteoblasts might not be prostaglandin related. These results suggest that the NSAID effects on cell cycle arrest and cell death induction in osteoblasts may be one of the important mechanisms contributing to their suppressive effect on bone formation.

Animals↗
PubMed1999

Transfection-mediated cell-cycle signaling: considerations for transient transfection-based cell-cycle studies.

Transient transfection of recombinant genes into cells is a commonly used approach for analyzing cell-cycle- and/or apoptotic-related activities of cell-cycle control proteins. In this approach, information regarding the functional consequence of expressing a recombinant protein transiently is garnered by comparing against results obtained from cells which are transfected with either a control expression plasmid and/or with mutant expression plasmids. In general however, little attention is paid to whether the transfection procedure itself influences these experiments. Using the calcium phosphate transfection method, we show that the introduction of DNA into cells induces signaling of the cell-cycle control machinery. In Hela cells, a transient increase in G0/G1 cells is observed 8 h after transfection. Furthermore, the introduction of DNA into several cell lines induces apoptosis. Transfection-mediated apoptosis can be elicited through a p53-independent mechanism, suggesting the possible extrapolation to many tumor cell lines. Last, we show that due to a likely cell-cycle-specific entry of marker genes into the nucleus, a highly biased cell-cycle distribution is observed in successfully transfected cells at early times following transfection. The importance of these issues in the interpretation as well as the design of transient transfection-based cell-cycle experiments is discussed.

Cell Adhesion↗
PubMed1996

Individual nuclei differ in their sensitivity to the cytoplasmic inducers of DNA synthesis: implications for the origin of cell cycle variability.

Nuclei of multinucleate cells generally initiate DNA synthesis simultaneously, suggesting that the timing of DNA synthesis depends upon the appearance of a cytoplasmic signal. In contrast, intact nuclei from quiescent mammalian cells initiate DNA synthesis asynchronously in cell-free extracts of Xenopus eggs, despite the common environment. Here we show that the two nuclei of permeabilized binucleate cells enter DNA synthesis coordinately in egg extracts, as they do in vivo, with different pairs of nuclei initiating replication at different times. This indicates that the two nuclei of a binucleate cell are identical in their sensitivity to the inducers of DNA synthesis in egg extracts; this sensitivity varies in general between the nuclei of unrelated cells. The asynchrony of DNA synthesis shown by unrelated nuclei in egg extracts is therefore not an artifact of the cell-free system but a reflection of genuine differences preexisting within the intact cell. Evidence that these differences between nuclei are responsible for a substantial fraction of G1 variability in living cells is presented.

Animals↗
PubMed1994

Coumarin modulates the cell-cycle progression of an MTV-EJras cell line.

The effects of coumarin (1,2-benzopyrone) on ras oncogene expression during the cell cycle of an MTV-EJras cell line was determined by flow cytometry. ras oncogene expression in cells was induced by dexamethasone and increased fivefold during G1/G0 phase and threefold in S phase. Dexamethasone also increased the percentage of cells in S phase from 21% to 31%, compared to phosphate-buffered-saline-treated control cells (P < 0.01). Coumarin decreased the percentage of dexamethasone-treated cells in S phase from 31% to 19%, with a concomitant increase in the percentage of cells in G1/G0 compared to control levels. Coumarin also reduced ras expression (mean fluorescence) by 40% in G1/G0 phase and 30% in S phase. We conclude that coumarin significantly reduces the cell-cycle progression of MTV-EJras cells and decreases ras expression in both G1 and S phases. These findings suggest a novel approach to the selective control of proliferation of malignant cells.

Animals↗
PubMed1991

Kinetic aspects of Ki-67 antigen expression in a normal cell line.

Using a normal cell line derived from a human fetus, the disappearance and reappearance of the Ki-67-reactive antigen following modification of the cell cycle was observed and estimated immunohistologically. It was found that G1/G0 arrest induced by serum deprivation resulted in loss of the antigen in 24 h in all but a few (usually less than 10%) of cells. Return to normal medium and resumption of growth was accompanied by reappearance in 30 h. When entry into S-phase was prevented by desferrioxamine, reappearance of the antigen still occurred but only lasted for about 24 h. Inhibition of protein synthesis with cycloheximide also caused fading and eventual loss of immunostaining. In view of the ease with which this antigen becomes undetectable with cessation of protein synthesis and interruption of the cell cycle, we agree with those who advise caution in the use of Ki-67 to measure growth fraction in changeable cell populations such as tumours.

Blood↗
PubMed1991

Isolation of a population of transiently transfected quiescent and senescent cells by magnetic affinity cell sorting.

Rous sarcoma virus (RSV) and cytomegalovirus (CMV) promoters were tested for activity in proliferating and nonproliferating (quiescent or senescent) human embryo fibroblasts. These promoters were cloned upstream of the coding sequence for the Tac subunit of the interleukin 2 receptor, and activity was calculated from the fraction of Tac antigen positive cells detected in a coupled transient transfection/magnetic affinity cell sorting assay. Differences in promoter activities are substantial in quiescent cells: the efficiency of the RSV promoter is no greater than background whereas the CMV promoter is equally active in serum concentrations ranging from 0.5 to 20%. While both promoters are functional in growing cells (WI-38 and HeLa), the CMV promoter exhibits twofold greater activity. Surprisingly, in senescent cells both promoters exhibit the same degree of activity.

Avian Sarcoma Viruses↗
PubMed2002

Enhanced expression of insulin-like growth factor binding protein-3 sensitizes the growth inhibitory effect of anticancer drugs in gastric cancer cells.

Insulin-like growth factor (IGF)-I and -II are potent mitogens and their mitogenic effects are modulated by IGF binding proteins (IGFBPs). In this study, we evaluated whether the enhanced expression of IGFBP-3 may increase the sensitivity of human gastric cancer cells to the anticancer drugs. We further investigated the potential mechanism for the growth inhibitory effect of anticancer drug induced-IGFBP-3 expression. These IGFBP-3-expressing gastric cancer cells showed a lower proliferation rate than IGFBP-3-non-expressing cells. Treatment with anticancer drugs resulted in up-regulation of IGFBP-3 expression in IGFBP-3-expressing cells. Interestingly the anticancer drug-induced-growth inhibition was more evident in IGFBP-3-expressing cells causing the IGFBP-3 expressing cells but not the IGFBP-3 non-expressing cells to accumulate in the G1/G0 phase and induce apoptosis. The exogenous addition of IGFBP-3 inhibited the growth of IGFBP-3-non-expressing cells, causing them to undergo apoptosis. Our data suggest that IGFBP-3 may have an important role in the biology of gastric cancer cell growth and provides a potential marker for predicting the responsiveness to anticancer drugs.

Antineoplastic Agents↗
PubMed1998

Synchronization of normal human keratinocyte in culture: its application to the analysis of 1,25-dihydroxyvitamin D3 effects on cell cycle.

In this report, we applied the isoleucine-deprivation method to normal human keratinocytes in cultivation with serum-free MCDB153 medium. The growing cells were synchronized by transfer to MCDB153 medium prepared without isoleucine, and the degree of synchronization was analyzed by the cell cycle distribution and phosphorylation of retinoblastoma protein (pRB), one of the tumor suppressor gene products. 1,25-dihidroxyvitamin D3 (1,25(OH)2D3), a biologically active form of vitamin D3, is supposed to induce the differentiation and growth inhibition of human keratinocytes caused by cell cycle arrest. We examined its effect on cell cycle kinetics, especially progression from G1/G0 to S-phase, by using this synchronized system. We showed that 10(-6) M 1,25(OH)2D3 inhibited the progression from G1/G0 to S-phase strikingly in synchronized normal human keratinocytes.

Blotting, Northern↗
PubMed1999

Growth/cell cycle regulation of Sp1 phosphorylation.

Sp1 sites can mediate growth/cell cycle induction of dihydrofolate reductase in late G1 (Jensen, D. E., Black, A. R. Swick, A. G., and Azizkhan, J. C. (1997) J. Cell. Biochem. 67, 24-31). To investigate mechanisms underlying this induction, effects of serum stimulation on regulation of Sp1 were examined. In Balb/c 3T3 cells, serum stimulation did not affect Sp1 synthesis or the relative binding of Sp1 family members to DNA; however, it did result in a rapid, approximately 2-fold increase in Sp1 levels and an approximately 3-fold increase in specific Sp1 phosphorylation in mid-G1. In normal human diploid fibroblasts, serum stimulation also increased Sp1 phosphorylation in mid-G1 but did not affect Sp1 levels. Therefore, Sp1 phosphorylation is regulated in a growth/cell cycle-dependent manner which correlates temporally with induction of dihydrofolate reductase transcription. Further studies revealed a kinase activity specifically associated with Sp1 in a growth-regulated manner. This activity is distinct from purified kinases previously shown to phosphorylate Sp1 in vitro and phosphorylates Sp1 between amino acids 612 and 678 in its C terminus, a region also phosphorylated in mid-G1 in vivo. Therefore, this study indicates that phosphorylation of the C terminus of Sp1 may play a role in the cell cycle regulation of its transcriptional activity.

3T3 Cells↗
PubMed1991

Cell cycle-dependent expression of CD4 antigen in a monocytoid cell line.

The CD4 molecule has several biological functions, physiologically as a receptor for major histocompatibility complex class II molecules on antigen-presenting cells, and pathologically as a receptor for human immunodeficiency virus (HIV) by its binding to the HIV envelope glycoprotein gp 120. The frequency of CD4+ cells has been shown to correlate positively with both susceptibility and cytopathogenic effect by HIV. To determine if CD4 expression varied during the cell cycle, a CD4-expressing monocytoid cell line, U 937 clone 16, was synchronized with regard to cell growth. The CD4 antigen was analysed with regard to expression, density and rate of reappearance after treatment with trypsin, during the different phases of the cell cycle. The CD4 reappearance rate was found to be maximal during the S phase. This was followed by an increased expression and density in the late S/G2 phase. Thus a cell cycle-dependent expression of CD4 molecules on the cell surface was observed.

CD4 Antigens↗
PubMed2001

Polyomavirus small t antigen prevents retinoic acid-induced retinoblastoma protein hypophosphorylation and redirects retinoic acid-induced G0 arrest and differentiation to apoptosis.

Polyomavirus small t antigen (ST) impedes late features of retinoic acid (RA)-induced HL-60 myeloid differentiation as well as growth arrest, causing apoptosis instead. HL-60 cells were stably transfected with ST. ST slowed the cell cycle, retarding G2/M in particular. Treated with RA, the ST transfectants continued to proliferate and underwent apoptosis. ST also impeded the normally RA-induced hypophosphorylation of the retinoblastoma tumor suppressor protein consistent with failure of the cells to arrest growth. The RA-treated transfectants expressed CD11b, an early cell surface differentiation marker, but inducible oxidative metabolism, a later and more mature functional differentiation marker, was largely inhibited. Instead, the cells underwent apoptosis. ST affected significant known components of RA signaling that result in G0 growth arrest and differentiation in wild-type HL-60. ST increased the basal amount of activated ERK2, which normally increases when wild-type cells are treated with RA. ST caused increased RARalpha expression, which is normally down regulated in RA-treated wild-type cells. The effects of ST on RA-induced myeloid differentiation did not extend to monocytic differentiation and G0 arrest induced by 1,25-dihydroxy vitamin D3, whose receptor is also a member of the steroid-thyroid hormone superfamily. In this case, ST abolished the usually induced G0 arrest and retarded, but did not block, differentiation without inducing apoptosis, thus uncoupling growth arrest and differentiation. In sum, the data show that ST disrupted the normal RA-induced program of G0 arrest and differentiation, causing the cells to abort differentiation and undergo apoptosis.

Antigens, Viral, Tumor↗
PubMed1996

Cell cycle arrest in G0/G1 phase by contact inhibition and TGF-beta 1 in mink Mv1Lu lung epithelial cells.

We postulated that contact inhibition and transforming growth factor (TGF)-beta 1 may target the same molecules to negatively regulate the Mv1Lu cell cycle in G0/G1. Both contact inhibition and TGF-beta 1 suppressed the expression of a 45-kDa protein (p45); cyclins D2 and B1; cyclin-dependent protein kinase (Cdk)-4, Cdc-2, and Cdc-2-associated activity; and the phosphorylation of retinoblastoma tumor-suppressor protein (pRb) but did not affect the expression of cyclins D1, E, and A or the expression of Cdk-2 and Cdk-5. Expression of p45 reappeared 12 h after release from contact inhibition and 6-8 h after release from TGF-beta 1, while TGF-beta 1 prevented release from contact inhibition and maintained suppression of both p45 and cyclin D2. Additionally, cyclin D2 phosphorylation and its associated kinase activity were strongly inhibited by contact inhibition and TGF-beta 1. Thus suppression of p45, cyclin D2/Cdk-4, and cyclin B1/Cdc-2 expression and/or activities is targeted both by contact inhibition and by TGF-beta 1 and may define common mechanisms through which these negative growth signals are integrated.

Animals↗
PubMed2002

Differential expression of receptors for Shiga and Cholera toxin is regulated by the cell cycle.

Cholera and Shiga toxin bind to the cell surface via glycolipid receptors GM1 and Gb3, respectively. Surprisingly, the majority of Vero cells from a non-synchronized population bind either Cholera or Shiga toxin but not both toxins. The hypothesis that the differential expression of toxin receptors is regulated by the cell cycle was tested. We find that Cholera toxin binds preferentially in G0/G1, with little binding through S-phase to telophase, whereas Shiga toxin binds maximally through G2 to telophase but does not bind during G0/G1 and S-phase. The changes result from the corresponding changes in Gb3 and GM1 synthesis, not from variations of receptor transport to the cell surface. The changes do not reflect competition of Gb3 and GM1 synthesis for lactosylceramide. Cells as diverse as Vero cells, PC12 cells and astrocytes show the same cell-cycle-dependent regulation of glycosphingolipid receptors, suggesting that this novel phenomenon is based on a conserved regulatory mechanism.

Animals↗
PubMed2001

Biological methods for cell-cycle synchronization of mammalian cells.

Understanding the molecular and biochemical basis of cellular growth and division involves the investigation of regulatory events that most often occur in a cell-cycle phase-dependent fashion. Studies examining cell-cycle regulatory mechanisms and progression invariably require cell-cycle synchronization of cell populations. Thus, many methods have been established to synchronize cells at specific phases of the cell cycle. Several of the common methods involve pharmacological agents, which act at various points throughout the cell cycle. Because of adverse cellular perturbations resulting from many of the synchronizing drugs used, other synchrony methods that involve less perturbation of biological systems, such as serum deprivation, contact inhibition, and centrifugal elutriation have a significant advantage. The advantages and disadvantages of these cell synchronization methods are discussed in this review.

Animals↗
PubMed2001

The late effects of proton irradiation on cell growth, cell cycle arrest and apoptosis in a human melanoma cell line.

The aim of this work is the in vitro study of the late effects of single proton irradiation on HTB63 human melanoma cell growth, cell cycle and cell death. The experimental conditions were focused on analyzing the effects of irradiation on the periphery of tumour that can be, in clinical practice, close to critical organs. Confluent cell monolayers were irradiated with single doses ranging from 1 - 20 Gy, using proton beams having an energy of 22.6 MeV at the target. Antiproliferative effect of protons, cell cycle analysis and initiation of cell death, were followed 48 hours after irradiation. The inhibition of melanoma cell growth was observed, especially after single application of 12 and 16 Gy. Cell cycle analysis and cell viability have shown the G2/M and G1/G0 arrest of irradiated cells correlating with the increase of the applied dose. The flow cytometric analysis has shown presence of apoptotic nuclei. These data demonstrate that irradiation with protons, under the chosen experimental conditions, have significant effects on melanoma cell growth inhibition being dose dependent, G2/M cell cycle arrest and appearance of apoptotic nuclei, even 48 hours after irradiation. The results obtained may help the understanding of the relationship between cell proliferation, death and cell cycle regulation of melanomas after proton irradiation.

Apoptosis↗
PubMed1991

Cell-cycle-related staining patterns of anti-proliferating cell nuclear antigen monoclonal antibodies. Comparison with BrdUrd labeling and Ki-67 staining.

Monoclonal antibodies (MAbs) to nuclear antigens are increasingly used as tools to obtain valuable information concerning the proliferative characteristics of various types of cancer. Prerequisite for the application of these MAbs in surgical pathology is establishment of the level of expression and/or cellular distribution of the antigens in relation to distinct cell-cycle compartments. In this study the topologic distribution of proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase delta, as recognized by human autoantiserum (AK) and two recently developed MAbs (19A2 and 19F4), was evaluated. Using cultured human cancer cells as a model system, and providing optimal fixation/permeation procedures are applied, these antibodies display a high affinity for PCNA bound to nuclear replicon clusters, resulting in distinct granular staining patterns. A more diffuse nucleoplasmic PCNA staining was mainly restricted to non-S-phase cells; in methanol-fixed cells, staining intensity of this form relative to the replicon-bound form appeared higher after staining with 19A2 than with 19F4 or AK. Comparing PCNA expression (detected with 19A2) with the expression of the Ki-67 antigen, PCNA-negative cells are also Ki-67 negative. In MCF-7 human breast cancer cells treated with 10(-6) mol/l (molar) tamoxifen, the fraction of nuclei showing replication patterns decreased from 42% to 8% within 8 days, but PCNA and Ki-67 antigens remained detectable in most cells during this interval, indicating a relatively slow decrease of antigen expression in cells that have entered a quiescent state. Treatment of MCF-7 cells with 10(-6) mol/l methotrexate resulted in a rapid accumulation of cells with an early S-phase DNA content; PCNA replication patterns showing a frequency distribution reflecting this DNA content were observed up to 48 hours after treatment. This indicates that the presence of replication patterns as visualized with anti-PCNAs is not a measure of replicative activity per se. It is concluded that, providing nuclear non-S-phase PCNA staining is faint relative to staining of replicon clusters, anti-PCNA antibodies may be excellent markers to detect in situ cells with S-phase DNA contents.

Antibodies, Monoclonal↗