Growth arrest of melanoma cells is differentially regulated by contact inhibition and serum deprivation.

Both growth-factor deprivation and contact inhibition suppress cell growth; however, the mechanisms by which they inhibit cell proliferation may not be identical. The function of antiproliferative genes and the induction of programmed cell death are among the potential differences between these growth-arrest mechanisms. Specifically, an inverse relation between the expression of cyclin-dependent kinase inhibitors (CDKIs) and the susceptibility to apoptosis has been reported. To test this relation, we examined the features of growth arrest in a canine melanoma cell line, TLM1. Both contact inhibition and serum deprivation halted cell-cycle progression of TLM1 cells in the G1 phase. Prolonged growth arrest of the cells without restimulation resulted in apoptosis; conversely, the cells reentered the cell cycle after release from contact inhibition or on restimulation with serum. Cell-to-cell contact, but not serum deprivation, led to the expression of p53 and p21/Waf-1. The expression of p21/Waf-1 did not prevent apoptosis. Moreover, the ectopic overexpression of CDKIs increased apoptosis. These results support the premise that growth arrest induced by contact inhibition and serum deprivation are mediated through distinct mechanisms. Furthermore, CDKIs are not universal inhibitors of apoptosis, and in some cases, they may initiate or enhance the apoptotic program.     

Growth arrest in G1 protects against oxygen-induced DNA damage and cell death

Although oxygen is required for normal aerobic respiration, hyperoxia (95% O(2)/5% CO(2)) damages DNA, inhibits proliferation in G1, S and G2 phases of the cell cycle, and induces necrosis. The current study examines whether growth arrest in G1 protects pulmonary epithelial cells from oxidative DNA damage and cell death. Mv1Lu pulmonary adenocarcinoma cells were chosen for studies because hyperoxia inhibits their proliferation in S and G2 phase, while they can be induced to arrest in G1 by altering culture conditions. Hyperoxia inhibited proliferation, increased intracellular redox, and rapidly reduced clonogenic survival. In contrast, Mv1Lu cells treated with transforming growth factor (TGF)-beta1, deprived of serum or grown to confluency, arrested and remained predominantly in G1 even during exposure. Growth arrest in G1 significantly enhanced clonogenic survival by 10-50-fold. Enhanced survival was not due to reduction in the intracellular redox-state of the cells, but instead was associated with reduced DNA strand breaks and p53 expression. Our findings suggest that the protective effects of G1 is mediated not simply by a reduction in intracellular ROS, but rather through an enhanced ability to limit or rapidly recognize and repair damaged DNA.     

Cleistanthin B causes G1 arrest and induces apoptosis in mammalian cells

Cleistanthin B is a potential anticancer agent isolated from the tropical plant Cleistanthus collinus. We have previously shown that cleistanthin B is clastogenic and induces micronuclei formation and chromosomal aberrations. We now show that this compound inhibits DNA synthesis in Chinese hamster ovary (CHO) cells and induces apoptosis in cervical carcinoma (SiHa) cells. Flow cytometric analysis of cleistanthin treated CHO cells revealed that they were blocked in G1. Cervical carcinoma (SiHa) cells exposed to cleistanthin B shrank, rounded up and had condensed chromatin and fragmented nuclei. DNA isolated from cleistanthin treated cells exhibited the characteristic apoptotic ladder when electrophoresed in agarose gels. These results were confirmed by flow cytometry. Etoposide, a structurally similar compound also induced apoptosis in these cells although with a difference. Etoposide induced apoptosis after permitting cells to enter into S phase, while cleistanthin B stopped entry of cells into S phase and subsequently drove them to apoptosis.     

Polycystin-1 induced apoptosis and cell cycle arrest in G0/G1 phase in cancer cells

Studies have shown that polycystin-1, encoded by PKD1, the major ADPKD, may have a central role in regulating both apoptosis and proliferation, which could prevent the malignant transformation of affected cells. However, as a putative tumor suppressor, direct studies on the possibility that polycystin-1 may play a role in cancer cells' biological properties have not yet been reported. We have demonstrated that the apoptosis of cancer cells was induced by overexpression of polycystin-1. After transfection with polycystin-1, three cancer cell lines, HepG2, A549, and SW480, showed significantly increased apoptosis compared with the respective control groups. This was accompanied by cell cycle arrest at G(0)/G(1) phase, whereas cell proliferation was not significantly affected. Overexpression of polycystin-1 induces apoptosis in cancer cells, at least partially, through Wnt and a caspase-dependent pathway.     

Ganoderic acid Me induces G1 arrest in wild-type p53 human tumor cells while G1/S transition arrest in p53-null cells

The mechanism of cell cycle arrest of tumor cells induced by ganoderic acid Me (GA-Me) is not understood. In this work, GA-Me was found to possess remarkable cytotoxicity on highly metastatic lung carcinoma 95-D cell line in both dose- and time-dependent manners. The effect of GA-Me on cell cycle arrest was found in 95-D, p53-null lung cancer cells H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? human colon cancer cells. To obtain an insight into the role of p53 in cell cycle arrest by GA-Me, 95-D, H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? cells were used for further investigation. GA-Me arrested cell cycle at G₁ phase in 95-D and HCT-116 p53^+/+ cells while S phase or G₁/S transition arrest in H1299 and HCT-116 p53^?/? cells. The results suggested that p53 may be a target of GA-Me, and it may be looked at as a new promising candidate for the treatment of carcinoma cells.     

Growth stimulation of rous sarcoma virus-transformed BHK cells by biotin and serum lipids

Biotin or a serum lipid extract stimulated proliferation of G1 arrested Rous sarcoma virus-transformed BHK cells in modified Eagle's MEM (BM). The cells could be maintained continuously in BM plus biotin (BMB), but not in BM plus serum lipid extract (BM X L). Avidin inhibited growth stimulation when added to BMB, but did not inhibit growth when added to BM X L. 14C-acetate incorporation into total cellular lipids was stimulated in BMB, but not in BM. Thin-layer chromatography of the labeled cellular lipid extract indicated that relatively large amounts of 14C-acetate were incorporated into phosphatidylserine and little into the other major phospholipids. In the neutral lipids, the largest amount of incorporation was in cholesterol. G1 arrested cells multiplied rapidly in BM supplemented with dialyzed serum (BM X DS), but they did not multiply in BM with delipidized serum (BM X DLS). The addition of biotin or serum lipid extract to BM X DLS stimulated growth. Growth stimulation in BM X DLS by biotin was inhibited by avidin, but avidin had no effect on growth stimulation by serum lipid extract. Biotin stimulated additional multiplication in BM X DS and avidin inhibited this additional growth stimulation. These results suggest that growth stimulation requires lipids supplied by serum lipids or by de novo synthesis stimulated by biotin. In the absence of serum, the stimulation of the synthesis of growth factor(s) by biotin are also required for continuous multiplication.     

IgG production kinetics in serum free media

Summary A murine hybridoma (455) was cultured in four different serum free media formulations, and a newborn calf serum supplemented medium was used as a basis of comparison. The serum supplemented medium supports a higher cell growth rate and results in a higher IgG titer. However, the antibody secretion rate on a per cell basis is higher in the serum free media, indicating that serum could be inhibitory to antibody secretion. The results identify the possibility of a least eliminating serum during the monoclonal antibody production phase.     

Diltiazem inhibits transferrin receptor expression and causes G1 arrest in normal and neoplastic T cells.

Transferrin receptor expression is essential for the proliferation of both normal and malignant T cells. While transferrin receptor expression in normal T cells is tightly coupled to interleukin-2 receptor expression, transferrin receptor expression in malignant cells is usually constitutive and is released from this constraint. Temporally, the appearance of these membrane receptors is preceded by changes in the expression of the proto-oncogenes c-myc and c-myb. In addition, although an increase in the level of intracellular free calcium occurs early in the sequence of T-cell activation, the activation events dependent on this calcium flux have not been resolved. In the present study we report that diltiazem, an ion channel-blocking agent that inhibits calcium influx, arrested the growth in vitro of both normal and malignant human T cells in the G1 phase of the cell cycle. However, diltiazem did not inhibit the expression of c-myc or interleukin-2 receptor mRNA and protein in normal mitogen-activated T cells or the constitutive expression of c-myc and c-myb mRNA in malignant T cells (T acute lymphoblastic leukemia cells). In contrast, diltiazem prevented the induction of transferrin receptor (mRNA and protein) in normal T cells and caused a progressive loss of transferrin receptor (mRNA and protein) in malignant T cells. These data demonstrate that diltiazem can dissociate several growth-related processes normally occurring in G1 and thereby disrupt the biochemical cascade leading to cell proliferation.     

Growth of Ibaraki virus in suspension culture of HmLu-1 cells.

The established hamster lung cell line, HmLu-1 cells could grow in a suspended state. The initial cell count, 40 X 10(4)/ml, increased to 200 X 10(4)/ml on the 4th day of culture. The suspension culture of HmLu-1 cells was proved satisfactory for propagation of Ibaraki virus. The viral titer reached a maximum of 10(6.75) TCID50/0.1 ml. The input multiplicity ranging from 0.003 to 3.0 exerted no influence on the final yield of the virus. The optimal pH value of initial culture ranged from 6.8 to 7.6. In comparison of virus yield per cell among the suspension culture and two methods of monolayer culture in stationary and rolling condition, there was no noticeable difference in it among the three methods. The cell population per unit volume was the largest and, therefore, virus titer in the culture fluid the highest in the suspension culture of the three methods.     

Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells.

Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of chicken DT40 cells disrupted in their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27(Kip1). Further more, we found that Cdh1(-/-) cells fail to maintain DNA damage-induced G2 arrest and that Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest.     

Strategic cell-cycle regulatory features that provide mammalian cells with tunable G1 length and reversible G1 arrest

Transitions between consecutive phases of the eukaryotic cell cycle are driven by the catalytic activity of selected sets of cyclin-dependent kinases (Cdks). Yet, their occurrence and precise timing is tightly scheduled by a variety of means including Cdk association with inhibitory/adaptor proteins (CKIs). Here we focus on the regulation of G1-phase duration by the end of which cells of multicelled organisms must decide whether to enter S phase or halt, and eventually then, differentiate, senesce or die to obey the homeostatic rules of their host. In mammalian cells, entry in and progression through G1 phase involve sequential phosphorylation and inactivation of the retinoblastoma Rb proteins, first, by cyclin D-Cdk4,6 with the help of CKIs of the Cip/Kip family and, next, by the cyclin E-Cdk2 complexes that are negatively regulated by Cip/Kip proteins. Using a dynamical modeling approach, we show that the very way how the Rb and Cip/Kip regulatory modules interact differentially with cyclin D-Cdk4,6 and cyclin E-Cdk2 provides to mammalian cells a powerful means to achieve an exquisitely-sensitive control of G1-phase duration and fully reversible G1 arrests. Consistently, corruption of either one of these two modules precludes G1 phase elongation and is able to convert G1 arrests from reversible to irreversible. This study unveils fundamental design principles of mammalian G1-phase regulation that are likely to confer to mammalian cells the ability to faithfully control the occurrence and timing of their division process in various conditions.     

Recovery of Saccharomyces cerevisiae mating-type a cells from G1 arrest by alpha factor.

Mating-type a cells of the yeast Saccharomyces cerevisiae that had been specifically arrested in the G1 phase of the cell cycle by alpha factor, an oligopeptide pheromone made by alpha cells, recovered and resumed cell division after a period of inhibition which was dependent on the concentration of alpha factor used. These treated a cells were more resistant to alpha factor than untreated a cells, but lost their resistance upon further cell division. However, cells arrested for 6 h were no more resistant to alpha factor than cells arrested for only 2.5 h. Mating-type a strains could inactivate or remove alpha factor from the culture fluid, but two a sterile (nonmating) mutants and an a/alpha diploid strain could not. These results suggest that a cells have a mechanism, which may involve uptake or inactivation of alpha factor, for recovering from alpha factor arrest. However, the results do not distinguish between a recovery mechanism which is constitutive and one which is induced by alpha factor. The loss of alpha factor activity during recovery appeared to be primarily cell contact mediated, although an extracellular, diffusible inhibitor of alpha factor that is labile or that functions stoichiometrically could not be ruled out.     

Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells.

Lactoferrin inhibits cell proliferation and suppresses tumor growth in vivo. However, the molecular mechanisms underlying these effects remain unknown. In this in vitro study, we demonstrate that treatment of breast carcinoma cells MDA-MB-231 with human lactoferrin induces growth arrest at the G1 to S transition of the cell cycle. This G1 arrest is associated with a dramatic decrease in the protein levels of Cdk2 and cyclin E correlated with an inhibition of the Cdk2 kinase activity. Cdk4 activity is also significantly decreased in the treated cells and is accompanied by an increased expression of the Cdk inhibitor p21(CIP1). Furthermore, we show that lactoferrin maintains the cell cycle progression regulator retinoblastoma protein pRb in a hypophosphorylated form. Additional experiments with synchronized cells by serum depletion confirm the anti-proliferative activity of human lactoferrin. These effects of lactoferrin occur through a p53-independent mechanism both in MDA-MB-231 cells and other epithelial cell lines such as HBL-100, MCF-7, and HT-29. These findings demonstrate that lactoferrin induces growth arrest by modulating the expression and the activity of key G1 regulatory proteins.     

Growth arrest of epithelial cells during measles virus infection is caused by upregulation of interferon regulatory factor 1

Natural infection with measles virus (MeV) is initiated when the virus reaches epithelial cells in the respiratory tract, oropharynx, or conjunctivae. Human epithelial cells infected with MeV frequently show growth suppression. In this study, we investigated the possible mechanisms for this suppression. The bronchiolar epithelial cell A549 showed growth arrest in G(0)/G(1) following MeV infection or treatment with gamma interferon (IFN-gamma). IFN regulatory factor-1 (IRF-1) was upregulated during MeV infection, although A549 did not produce IFN-gamma. Cells of the cervical squamous cell line SiHa persistently infected with various strains of MeV displayed slower growth than uninfected SiHa cells, although the growth rates varied depending on the MeV strain. Transfection of antisense-oriented IRF-1 cDNA released the MeV-infected SiHa cells from growth suppression. Although these infected cells did not produce IFN-gamma and suppressed IFN-alpha/beta-induced Jak1 phosphorylation, Jak1 was constitutively phosphorylated. The growth rates negatively correlated with levels of both IRF-1 expression and constitutively phosphorylated Jak1. These results indicate that MeV upregulates IRF-1 in a manner that is independent of IFN but dependent on the JAK/STAT pathway. This induction of IRF-1 appears to suppress cell growth, although the extent seems to vary among MeV strains.     

Overexpression of Hp95 induces G1 phase arrest in confluent HeLa cells

Xp95, a protein recently identified in Xenopus laevis, is potentially involved in progesterone-induced Xenopus oocyte maturation. In this study, we cloned a human homologue of Xp95, designated Hp95, and examined the effect of its overexpression on the growth properties of human malignant HeLa cells which have lost the contact inhibition of cell proliferation. We observed that although HeLa cells did not undergo G1 phase arrest at any stage after confluence, they were able to downregulate their G1 phase CDK activities in response to confluence. When Hp95 was overexpressed in HeLa cells by transfection with a constitutive or an inducible expression vector containing a full-length Hp95 transgene, HeLa cells became able to undergo G1 phase arrest and form a monolayer culture after confluence. However, the G1 phase CDK activities in these Hp95 overexpressing cells were not inhibited further as compared to control cells after confluence. These results indicate that the defects in HeLa cells that cause the loss of contact inhibition of cell proliferation are in components downstream of the G1 phase CDKs and that overexpression of Hp95 counteracts some of these defects.     

Smad7 induces G0/G1 cell cycle arrest in mesenchymal cells by inhibiting the expression of G1 cyclins

The major Smad pathways serve in regulating the expression of genes downstream of TGFbeta signals. In this study, we examined the effects of sustained Smad7 expression in cultured cells. Interestingly, Smad7 caused various mesenchymal cells, including NIH3T3 fibroblast and ST2 bone-marrow stromal cells, to undergo a marked morphological alteration into a flattened cell shape, but kept them alive for as long as 60 days. Furthermore, Smad7 arrested the proliferation of the cells even before they reached confluence. These cells became quiescent in G0/G1 phase and accumulated a hypophosphorylated form of retinoblastoma. The cytostatic effect of Smad7 was closely associated with a preceding decrease in the levels of G1 cyclins, such as cyclin D1 and cyclin E. Accordingly, ectopic cyclin E was able to overcome the Smad7-induced arrest of proliferation. These results indicate that Smad7 functions upstream of G1 cyclins and suggest a novel role for Smad7 as an antiproliferative factor. In contrast to the growth of mesenchymal cells, that of epithelial cells was little susceptible to Smad7. The present findings raise the possibility that a link between Smad7 and the G1 to S phase transition may also contribute to the cell cycle control by certain Smad7-inducing stimuli in a cell-type-dependent fashion.