Effect of staurosporine on MOLT-4 cell progression through G2 and on cytokinesis

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G₁, most virus transformed or tumor cells are unaffected in G₁ but arrest in G₂ phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nucleolar p120 protein, or protein reactive with Ki-67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT-4 cells in the presence of 0.1 μM SSP.MOLT-4 cells did not arrest in G₁ or G₂ phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G₁ phase at higher DNA ploidy (tetraploidy; G_1T), and then progressed through S_T (rereplication) into G_2T and M_T. The rates of entrance to G₂ and G_2T were essentially identical, indicating that the rates of cell progression through S and S_T as well as through G₂ and G_2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 μM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT-4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis. © 1994 Wiley-Liss, Inc.     

Increased labelling of polyphosphoinositide in chemically transformed cell line C3H10T1/2 CL8

The effect of malignant transformation of cells on phosphatidylinositol metabolism was investigated using C3H10T1/2 cells and its chemically transformed cell line, MCA CL-16 cells. We found that incorporation of [³²P]P_i into polyphosphoinositide was greatly increased in the transformed cells. A similar tendency was observed when myo-[2-³H]inositol was used as a labelling reagent. It is also observed that influx of labelled inorganic phosphate is enhanced 2-fold by the cell transformation. Therefore, promotion of polyphosphoinositide labelling in the transformed cell might be caused not only by the enhanced metabolism of phosphatidylinositol but also by the increased membrane permeability for radioactive labelling reagents.     

Opposing roles for CD34 in B16 melanoma tumor growth alter early stage vasculature and late stage immune cell infiltration

Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34(-/-) mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34(-/-) mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34(-/-) mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.     

Cell cycle suspension: A novel process lurking in G2 arrest

Cell cycle checkpoint is a self-protective mechanism for cells to monitor genome integrity and ensure the high-fidelity transmission of genetic information to daughter cells. Insufficient function of cell cycle checkpoints has been demonstrated to partially account for tumor initiation, promotion and progression. In the ten melanoma cell lines that we tested in preliminary experiments, two human uveal melanoma cell lines, 92-1 and OCM-1, were found to be significantly different in terms of radiosensitivity but similar in DNA repair ability. Evident G₂ arrest was induced in both cell types and the maximum was reached at 16 h after irradiation regardless of X-rays or high-LET carbon beams. OCM-1 cells overrode the G₂ arrest and reentered the cell cycle right after reaching the maximum, whereas 92-1 could not. Upon 10 Gy of radiation, the cell cycle of 92-1 was suspended and remained unchanged for up to 5 d. The cell cycle suspension is a unique process lurking in G₂ arrest and related to cellular radiosensitivity. Its induction is dose-dependent and there is a dose threshold for it. The degradation of Cyclin B1 has been found related to the cell cycle suspension though, the mechanism of cell cycle suspension is still under investigation. Basing on our knowledge, this is the first report on cell cycle suspension and we present here a de novo mechanism to cellular radiosensitivity. Further clarification of the mechanism underlying cell cycle suspension is believed to be of significance in tumor radiosensitization or even direct tumor control.     

Effect of magnesium on the growth and cell cycle of transformed and non-transformed epithelial rat liver cells in vitro

The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epithelial-like rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 × the concentration in the regular RPMI 1640 media (100mg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G₁ and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G₁ with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G₁ nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics.     

RELATIONSHIP OF NUTRITIONAL FACTORS TO IN VITRO TUMOR CELL GROWTH AND CYTOTOXICITY PRODUCED BY CYTOSINE ARABINOSIDE

The in vitro relationship between nutritional factors, proliferative status of tumor cells, and the cytotoxic action of cytosine arabinoside (ara-C) was investigated. The reduction in the concentration of only one essential amino acid, L-isoleucine, in the growth medium of A(T₁)Cl-3 hamster fibrosarcoma cells decreased DNA synthesis in this cell population and slowed the rate of progression of G₁ phase cells into S phase of the cell cycle. The complete omission of isoleucine from the growth medium blocked the progression of G₁ phase cells into S phase and prevented the cytotoxic action of ara-C. The addition of isoleucine to the isoleucine-deprived cells permitted these cells to enter the S phase and restored their sensitivity to the cytotoxic action of ara-C. When G₁ phase cells were placed in a medium containing reduced levels of all the amino acids and vitamins there was a prolongation of the G₁ phase. Since medium with low levels of amino acids produced a delay in the entry of G₁ phase cells into the S phase, the time interval in which these cells were most sensitive to the cytotoxic action of ara-C was different for G₁ phase cells placed in medium with adequate levels of all the amino acids. These in vitro data indicate that nutritional factors can markedly effect the proliferation of tumor cells and the cytotoxic action of ara-C.     

Connexins and cancer

The hypothesis, that gap junctional intercellular communication plays a key role in carcinogenesis and more generally in growth control was formulated nearly 40 years ago. From this time, data accumulated, showing that this type of communication is frequently decreased or absent in cells treated with tumor promoting agents, among transformed cells or between transformed/tumor cells and normal cells. This observation has been made on various cell types and whatever their tissue and species origins, by using in vitro and in vivo models. It led to the general assumption that the inhibition of gap junctional intercellular communication may play a role in carcinogenesis at two levels: (1) during tumor promotion by favoring the clonal expansion of initiated cells and (2) after the phenotypic transformation of cells by preventing the diffusion of putative "normalizing" factors between tumor cells and surrounding normal cells. During the past decade, the discovery that gap junction proteins, the connexins (Cx), may act as tumour suppressors, by reverting the phenotype of transformed cells confirmed the idea that their lack of function would be actively involved in carcinogenesis. However, we still do not know precisely what are the molecular processes that gap junctional intercellular communication may regulate and still do have very few data concerning the gap junction situation in human cancers. All these aspects are presented from an historical point of view and discussed below.     

Flow cytometric BrdUrd-pulse-chase study of X-ray-induced alterations in cell cycle progression

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd)-pulse-chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X-rays and pulse-labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd-free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G₂, i.e. the fraction of BrdUrd-negative G₂ cells and the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays. The magnitude of the effects indicates that the cells irradiated in G₂ and in S are equally delayed. S phase transit of cells irradiated in S or in G₁ did not appear to be affected. There were apparent changes in flow of cells out of G₁, which could be explained by the delayed entry of G₂ cells into the compartment because of G₂ arrest. Thus, in asynchronous cells the method was able to detect G₂ delay in those cells irradiated in S and G₂ phases and demonstrate the absence of cell-cycle delays in other phases.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

Prolyl oligopeptidase (POP) is a post-proline cleaving enzyme, which is widely distributed in various organs, with high levels in the brain. In this study, we investigated the effects of a selective POP inhibitor, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746), on the growth of NB-1 human neuroblastoma cells. SUAM-14746 treatment for 24–72 h suppresses the growth of NB-1 cells without cell death in a dose-dependent manner (10–60 μM). Similar suppressive effects were observed with another POP inhibitor benzyloxycarbonyl-thioprolyl-thioprolinal. The SUAM-14746-induced growth inhibition in NB-1 cells was associated with pronounced G₀/G₁ arrest and reduced levels of phosphorylated retinoblastoma protein (pRb), cyclin E, and cyclin dependent kinase (CDK) 2, and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. SUAM-14746 also induced transient inhibition of S and G₂/M phase progression, which was correlated with retardation of the decrease in the levels of cyclins A and B. Moreover, RNAi-mediated knockdown of POP also led to inhibition of NB-1 cell growth and the effect was accompanied by G₀/G₁ arrest. These results indicate that POP is a part of the machinery that controls the cell cycle.     

TRIM16 inhibits neuroblastoma cell proliferation through cell cycle regulation and dynamic nuclear localization

Neuroblastoma is the most common solid tumor in childhood and represents 15% of all children’s cancer deaths. We have previously demonstrated that tripartite motif 16 (TRIM16), a member of the RING B-box coiled-coil (RBCC)/tripartite totif (TRIM) protein family, has significant effects on neuroblastoma proliferation and migration in vitro and tumorigenicity in vivo. However, the mechanism by which this putative tumor suppressor influences cell proliferation and tumorigenicity was undetermined. Here we show, for the first time, TRIM16’s striking pattern of expression and dynamic localization during cell cycle progression and neuroblastoma tumor development. In a tyrosine hydroxylase MYCN (TH-MYCN) neuroblastoma mouse model, immunohistochemical staining revealed strong nuclear TRIM16 expression in differentiating ganglia cells but not in the tumor-initiating cells. Furthermore in vitro studies clearly demonstrated that during G₁ cell cycle phase, TRIM16 protein expression is upregulated and shifts to the nucleus of cells. TRIM16 also plays a role in cell cycle progression through changes in Cyclin D1 and p27 expression. Importantly, using TRIM16 deletion mutants, an uncharacterized protein domain of TRIM16 was found to be required for both TRIM16’s growth inhibitory effects and its nuclear localization. Taken together, our data suggest that TRIM16 acts as a novel regulator of both neuroblastoma G₁/S progression and cell differentiation.     

Autocrine regulation of cell cycle progression in normal human keratinocytes

Summary Removal of competence factors insulin and pituitary extract from the culture medium, concomitant with the addition of picomolar concentrations of the late-G₁ inhibitor transforming growth factor-beta, effectively arrested cell cycle progression of normal human keratinocytes prior to their entry into the DNA synthesis phase; arrest continued for a minimum of 36 h following removal of unbound inhibitor and subsequent addition of factor-deficient medium. To demonstrate the reversibility of transforming growth factor-beta-induced arrest, two dissimilar cell populations were recruited to synthesize DNA in a predictable and reproducible manner; whereas the reinstatement of omitted competence factors induced noncycling cells to begin synthesizing DNA within 24 h, addition of keratinocyte-conditioned medium prompted an immediate progression of late-G₁ cells into S phase. Studies to determine the extent that autocrine signaling regulates cell cycle progression revealed that nontransformed keratinocytes produce an endogenous factor required for DNA replication and that production of this progression factor required competence factors insulin and pituitary extract. Keratinocyte progression factor recruited late-G₁ cells into S phase within 1–2 h, reversed transforming growth factor-beta-induced arrest in the presence of bound inhibitor, and elicited a calcium mobilization response consistent with receptor-mediated signaling. Hence, these studies demonstrate that G₁ progression of nontransformed keratinocytes into S phase requires an endogenous progression factor and suggest that this factor may direct G₁ progression by modulating the activity of a calcium-dependent kinase.     

Action of FR901228, a Novel Antitumor Bicyclic Depsipeptide Produced by Chromo-bacterium violaceum No. 968, on Ha-ras Transformed NIH3T3 Cells

FR901228, a novel antitumor antibiotic, reversed the transformed morphology of the Ha-ras transformants, Ras-1 cells, and inhibited their growth. The reduction of c-myc expression was observed in FR901228-treated Ras-1 cells by RNA dot-blot hybridization. This reduction of c-myc expression and morphological reversion of the transformed cells to normal were correlated with growth inhibition (G₀/G₁ arrest in cell cycle).     

Normal versus abnormal cell proliferation

The most recent findings on the molecular and cellular characterization of normal and abnormal cell proliferation are summarized. They include molecular spectroscopy, nucleic acid conformation, protein modifications, premature chromosome condensation, nuceoli changes, nuclear and cell morphometry, image analysis, flow microfluorimetry, and time-lapse cinematography. Biophysical and biochemical evidence in favor or against two cycles of chromatin condensation, followed by two abrupt random decondensations, per cell cycle are presented. Other biphasic changes at the molecular and cellular levels that favor the existence of two random transitions, or restriction points, per cell cycle are discussed. A comprehensive unitary model of the cell cycle is then outlined; this model is able to explain most findings on continuously dividing cells and on quiescent cells induced to proliferate. Within this analytical framework the physical-chemical and biological properties are given, in either normal or tumor cells, for the various types of “noncycling” cells that are here viewed as necessary steps in mammalian cell growth rather than separate states. The implications of the coupling of higher-order chromatin structure with cell geometry and growth, high in fibroblast-like cells but low in transformed cells, are also discussed. Molecular mechanisms likely responsible for the chromatin conformational changes occurring at the G₀→G₁, G₁→S, G₂→M transitions are finally discussed in terms of polyelectrolyte theory.     

Relationship of Cytoskeletal Filaments to Annular Gap Junction Expression in Human Adrenal Cortical Tumor Cells in Culture

In addition to the well-characterized surface gap junctions expressed at contact sites between cells, annular gap junction profiles have been localized within the cytoplasm of some cell populations. To study and characterize these annular profiles, gap junction protein type was demonstrated with Western blot and immunocytochemistry. The distribution of annular gap junctions and the relationships to cytoskeletal elements were demonstrated with immunocytochemical, transmission electron microscopic, or image analysis with confocal microscopy techniques. SW-13 adrenal cortical tumor cells expressed α₁gap junctions at areas of cell to cell contact. In addition, α₁gap junction annular profiles were seen within the cytoplasm. Actin and myosin II were found closely associated with these annular gap junctions, while no physical association between tubulin- or vimentin-containing fibers and gap junction protein could be established. Disruption of microfilaments with cytochalasin B treatment (10 μg/ml, 1 h) resulted in a decrease in the average number and an increase in the average size of annular gap junctions compared to control populations. The results are consistent with a role for cytoskeletal elements containing actin and myosin II in annular gap junction turnover.     

Cyclic nucleotides and cell growth

Growth induction in resting fibroblast cultures by serum or growth factors induces a fast, transient cGMP peak which may constitute the intracellular signal for growth. A similar cGMP peak occurs when 3T3 cells arrested at the restriction point or in G₀ by starvation for certain amino acids are induced for growth by readdition of the lacking nutrients. Both 3T3 and SV3T3 cells which are arrested randomly all around the cell cycle do not exhibit major changes in cyclic nucleotides after growth induction. Determination of intracellular cAMP and cGMP levels in normal and transformed fibroblasts under different growth conditions shows that the transition between growing and resting state (G₀ arrest) is accompanied and probably induced by characteristic changes in cAMP to cGMP ratios. cGMP is decreased 2-5-fold in resting as compared to growing cultures, and increased 10-20-fold in activated cultures 20 min after serum induction. No major cGMP change was observed in growing, confluent, or serum-activated cultures of transformed cells. Measurement of guanylcyclase under unphysiological conditions (2 mM Mn⁺⁺) in crude and purified membranes from 3T3 and SV3T3 cultures did not show increased enzyme activity in the transformed cells. Significant differences may only show up when synchronized cells pass through the restriction point in G₁ phase. As a hypothesis it is proposed that transformed cells have an activated guanylcyclase system or a relaxed cGMP-pleiotypic response mechanism at the restriction point of their cell cycle.     

Inhibition of neoplastic cell growth by quiescent cells is mediated by serum concentration and cAMP phosphodiesterase inhibitors

We have demonstrated that confluent monolayers of the mouse fibroblast cell line C3H/10T1/2 (10T1/2) have the ability to cause reversible growth inhibition of cocultured transformed cells. This was first demonstrated for de novo transformed cells and later extended to established cell lines of proven oncogenicity in vivo. This growth inhibition could be increased by growing the 10T1/2 cells to high density in increasing concentrations of serum or by elevating intracellular concentrations of cAMP using inhibitors of phosphodiesterase (PDE). These manipulations, which in cocultures of nontransformed and transformed cells caused complete inhibition of tumor cell growth, had no effect on growth rate or saturation density of either ceil type when cultured alone, demonstrating the cooperative nature of this phenomenon. This cooperation could not be produced by transfer of culture medium, demonstrating the requirement for intimate cell contact. Inhibition of the formation of transformed foci of cells in these mixed cultures was accompanied by a decrease in the incorporation of labeled thymidine into these cultures; the kinetics of this inhibition and recovery suggested a rapidly reversible effect on cell cycle transit times. The potent inhibitor of cAMP PDE, Ro 20-1724 induced dose dependent increases in intracellular cAMP in both nontransformed and in transformed cells. However, at a concentration of 10^?4 M Ro 20-1724, which inhibited tumor cell growth in mixed cultures, cAMP was elevated 30-fold in nontransformed versus only 3-fold in transformed cells. The inhibitory effects of PDE inhibitors on tumor growth have been extended to an in vivo model system, utilizing Lewis lung carcinoma cells growing as metastases in the lungs of C57B1 mice. In these mice, inoculated intravenously with a single cell suspension of Lewis lung cells, the formation of lung metastases was dramatically decreased by the twice daily administration of either isobutylmethylxanthine or Ro 20-1724; PDE inhibitors were shown to be active in vitro. The latter compound, which showed highest activity in vitro, was also substantially more potent in vivo as an inhibitor of lung tumor colony formation and doubled the life span of the tumor bearing animals. Cell cycle analysis of lung tumor colonies by the labeled mitosis method showed that both phosphodiesterase inhibitors caused a prolonged G₁ phase in the cell cycle but failed to influence other phases. Although detailed analysis of host tissues is not complete, prolonged treatment with these drugs caused no statistically significant weight loss or changes in counts of red or white blood cells indicating a selective growth inhibition of transformed cells at these doses. Studies to determine the mechanism of the cellular communication and the nature of the signal are in progress.     

In Vivo Fluorescence Imaging Reveals the Promotion of Mammary Tumorigenesis by Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are multipotent adult stem cells which are recruited to the tumor microenvironment (TME) and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.     

The cancer‐related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre‐osteoblast proliferation by affecting cell cycle progression in the G₁ phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G₁/S phase transition, and Runx2 is again up‐regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle‐regulated with respect to the G₁/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre‐established levels in a given cell type triggers one or more anti‐proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. J. Cell. Physiol. 228: 714–723, 2013. © 2012 Wiley Periodicals, Inc.