Cyclic AMP-dependent phosphorylation of cyclin D3-bound CDK4 determines the passage through the cell cycle restriction point in thyroid epithelial cells

According to current concepts, the cell cycle commitment after restriction (R) point passage requires the sustained stimulation by mitogens of the synthesis of labile d-type cyclins, which associate with cyclin-dependent kinase (CDK) 4/6 to phosphorylate pRb family proteins and sequester the CDK inhibitor p27kip1. In primary cultures of dog thyroid epithelial cells, the cAMP-dependent cell cycle induced by a sustained stimulation by thyrotropin or forskolin differs from growth factor mitogenic pathways, as cAMP does not upregulate d-type cyclins but increases p27 levels. Instead, cAMP induces the assembly of required cyclin D3-CDK4 complexes, which associate with nuclear p27. In this study, the arrest of forskolin stimulation rapidly slowed down the entry of dog thyrocytes into S phase and the phosphorylation of pRb family proteins. The pRb kinase activity, but not the formation, of the cyclin D3-CDK4-p27 complex was strongly reduced. Using two-dimensional gel electrophoresis, a phosphorylated form of CDK4 was separated. It appeared in response to forskolin and was bound to both cyclin D3 and p27, presumably reflecting the activating Thr-172 phosphorylation of CDK4. Upon forskolin withdrawal or after cycloheximide addition, this CDK4 phosphoform unexpectedly persisted in p27 complexes devoid of cyclin D3 but it disappeared from the more labile cyclin D3 complexes. These data demonstrate that the assembly of the cyclin D3-CDK4-p27 holoenzyme and the subsequent phosphorylation and activation of CDK4 depend on distinct cAMP actions. This provides a first example of a crucial regulation of CDK4 phosphorylation by a mitogenic cascade and a novel mechanism of cell cycle control at the R point.     

Cyclic AMP inhibits developmental regulation of Chlamydia trachomatis.

The effect of cyclic AMP (cAMP) on the chlamydial growth cycle was studied with Chlamydia trachomatis-infected HeLa cells. At concentrations of 1 mM, cAMP had a profound effect on the chlamydial developmental cycle, resulting in small, immature inclusions. Immunoblot analysis revealed the absence of elementary body (EB)-specific antigens in the cAMP-treated cells. This effect was observed only if cAMP was added within the first 12 h of incubation and continued thereafter. Its withdrawal at any time from the medium led to the reappearance of fully mature, infectious organisms. Analogs or breakdown products of cAMP exerted no inhibitory effect on chlamydial development. Intracellular inclusions from the cAMP-treated cells were unable to infect fresh HeLa monolayers, in contrast to the completely infectious nontreated inclusions. Protein profiles of the cAMP-treated organisms (at any time point) resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis very closely resembled reticulate bodies (RB) and did not possess characteristic EB-binding proteins. Collectively, these observations suggest an inhibitory role for cAMP at the RB stage of intracellular development. We also identified a cAMP receptor protein which is associated with RB and not with EB, further supporting a role for this system in the developmental regulation of chlamydiae.     

Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels

Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299) exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells.     

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.     

Cyclic AMP levels in temperature sensitive SV40 transformed cell lines

The cAMP levels of the 3T3 cell line and its transformed derivatives were determined. The level was found to be lower in transformed cell lines than in parental 3T3 and to decrease at confluence in all cell lines tested. SV40 transformed 3T3 cell lines which are temperature sensitive in growth control were found to have lower cAMP levels than 3T3, but these levels were not temperature dependent. Retransformation of these cell lines by murine sarcoma virus did not markedly affect their cAMP levels.     

Cyclic AMP independence of Escherichia coli protein phosphorylation

The effect of cyclic AMP on protein phosphorylation was analyzed comparatively in two strains of E.coli differing in their capacity to synthesize this nucleotide, one of them lacking the adenylate cyclase activity. The results obtained from both in vivo and in vitro experiments concurred in showing that the bacterial protein kinase activity is cAMP-independent.     

Lidocaine inhibits melanoma cell proliferation by regulating ERK phosphorylation

The melanoma is responsible for the majority of all skin cancer–related deaths worldwide. Evidence suggests that local anesthetics provide some benefit in the treatment of cancer via inhibition of cellular proliferation, invasion and migration. However, the potential antiproliferative effects of local anesthetics in the treatment of melanoma remain to be elucidated. In this study, we investigated the antiproliferative effects and underlying mechanism of the commonly used local anesthetic (lidocaine) on melanoma cells. A375 melanoma cells were treated by lidocaine or vemurafenib. Cell Counting Kit-8, histological staining, flow cytometric analysis, immunohistochemical staining, and Western blot analyses were carried out to test the effects of lidocaine and vemurafenib on A375 cells. BALB/C-nu/nu mice intraperitoneally injected with A375 cells were treated by lidocaine, and then tumor volume and weight were calculated. Lidocaine exhibited vemurafenib-like effects totally. Lidocaine inhibited A375 melanoma cell proliferation in a dose- and time-dependent manner and colony formation also showed a dose-dependent inhibition. Lidocaine treatment resulted in the arrest of cell-cycle progression in the G1 phase and inhibited Ki-67 expression in a dose-dependent manner. This effect was associated with inhibited extracellular signal–regulated kinase (ERK) phosphorylation. In vivo experiments revealed that intravenous injections of lidocaine suppressed tumor volume and weight. Lidocaine inhibits melanoma cell proliferation in a dose- and time-dependent manner via a mechanism that may involve inhibition of the ERK signaling pathway. Thus, lidocaine may provide some benefit for the treatment of melanoma.     

The regulation of cell proliferation by calcium and cyclic AMP

Calcium, in partnership with cyclic AMP, controls the proliferation of non-tumorigenic cells in vitro and in vivo. While it does not seem to be involved in the proliferative activation of cells such as hepatocytes (in vivo) or small lymphocytes (in vitro), it does control two later stages of prereplicative (G1) development. It must be one of the very many regulatory and permissive factors affecting early prereplicative development, because severe calcium deprivation reversibly arrests some types of cell early in the G1 phase of their growth-division cycle in vitro. However, calcium more specifically and much more often regulates a later (mid or late G1) stage of prereplicative development. Thus, regardless of its severity or the type of cell, calcium deprivation in vitro or in vivo reversibly stops proliferative development at that part of the G1 phase in which the cellular cyclic AMP content transiently rises and the synthesis of the four deoxyribonucleotides begins. The evidence points to calcium and the cyclic AMP surge being co-generators of the signal committing the cell to DNA synthesis. The evidence is best explained so far by the cyclic AMP surge causing a surge of calcium ions which combine with molecules of the multi-purpose, calcium-dependent, regulator protein calmodulin (CDR) somewhere between the cell surface and the cytosol. The resulting Ca-calmodulin complexes then stimulate many different (and possibly membrane-associated) enzymes such as protein kinases, one of which produces the DNA-synthetic initiator. Calcium has little or no influence on the proliferation of tumor cells. Some possible explanations of this very important loss of control are considered.     

Cyclic AMP induces terminal deoxynucleotidyl transferase in immature B cell leukemia lines

Terminal deoxynucleotidyl transferase (Tdt), a unique DNA polymerase found only in lymphoid cells, may be involved in the generation of immunoglobulin-combining site diversity. To study the actual metabolic function of the enzyme, we developed a system in which Tdt expression is induced under defined culture conditions. We found that pharmacologic agents that raise intracellular cyclic AMP levels, such as caffeine, induce a three- to 10-fold increase in enzyme biosynthesis rate and activity. This phenomenon is observed only in pre-B cell lines of human or murine origin.     

CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytes

INTRODUCTION/OBJECTIVES: Cell cycle progression is driven by the coordinated regulation of cyclin-dependent kinases (CDKs). In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G(1) phase. It is an on-going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. METHODS AND RESULTS: In this study, we have explored the role of CDK4 activity during G(1) progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1-induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2-Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. CONCLUSIONS: Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G(1) phase.     

Cyclic AMP decreases the phosphorylation state of myelin basic proteins in rat brain cell cultures

Previous work has suggested that myelin basic proteins are phosphorylated prior to their appearance in the myelin sheath (Ulmer, J. B. and Braun, P. E. (1984) Dev. Neurosci. 6, 345-355). In order to corroborate this finding we have examined the phosphorylation of myelin basic proteins in rat brain cell cultures containing 14-17% oligodendrocytes. Incorporation of 32P into the 14-, 17-, 18.5-, and 21.5-kDa myelin basic proteins was observed in cells incubated with 32P at 7, 14, and 21 days in culture. Myelin basic proteins in 14-day cells incorporated 32P linearly until at least 120 min after the addition of isotope. The apparent half-life of myelin basic protein phosphate groups was determined to be approximately 80 min in pulse-chase experiments. However, this value may be an overestimation due to the presence of significant levels of acid-soluble radioactivity in the cells throughout the chase period. The presence of dibutyryl cAMP or 8-bromo-cAMP in the incubation medium substantially inhibited the incorporation of 32P into the myelin basic proteins at all time points studied. The presence of dibutyryl cAMP in the chase medium in pulse-chase experiments resulted in an increase in the turnover rate of [32P] phosphate in the myelin basic proteins. These results indicate that cAMP decreases the phosphorylation state of myelin basic proteins in oligodendrocytes by inhibiting the phosphorylation and/or stimulating the dephosphorylation of myelin basic proteins.     

Cyclic AMP and the regulation of cholesterol metabolism.

Cyclic AMP has been implicated to a greater or lesser extent in the regulation of four key enzymes which interact to regulate intracellular cholesterol metabolism; HMG CoA reductase; ACAT; cholesteryl ester hydrolase; and cholesterol 7 alpha hydroxylase. The relationship between these enzymes and the sites where current evidence suggests that cyclic AMP may be involved are summarized in Fig. 3. Cholesterol 7 alpha hydroxylase controls the catabolism of cholesterol to bile acids in the liver, and thus its removal from the body via the bile, but does not have a major role in cholesterol metabolism in extrahepatic tissues. It is clear that cyclic AMP is able to influence the activity of this enzyme in liver sub-cellular fractions and isolated hepatocytes in vitro, and studies in our laboratory have shown that changes in Ca2+ fluxes within the cell may be important in its mechanism of action. Whether or not the cyclic nucleotide has a role regulating cholesterol 7 alpha hydroxylase activity in vivo, however, is not known. HMG CoA reductase is inactivated by phosphorylation both in vitro and in vivo, but although cyclic AMP and glucagon have been shown to inhibit the enzyme, cyclic AMP-dependent protein kinase is not directly involved. The exact mechanism by which the cyclic nucleotide influences the system remains unclear, but it may be related to activation of microsomal phosphatases. The activity of ACAT has been shown to be modulated by phosphorylation in a number of tissues in vitro, but the involvement of cyclic AMP has not been unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP in relation to proliferation of the epidermal cell: a new view.

Four agents known to increase the level of cellular cAMP by different means (cholera toxin, dibutyryl cAMP, methyl isobutyl xanthine and isoproterenol) increase the growth of colonies of cultured human epidermal cells and of keratinocytes derived from other stratified squamous epithelia. This effect is due to an increase in the overall rate of cell proliferation in the colonies. When added to cultures under hitherto optimum conditions for epidermal cell growth [in the presence of supporting 3T3 cells and epidermal growth factor (EGF)], most of the agents exert an effect of considerable magnitude, the toxin being the most potent. Since the toxin exerts an effect in the absence of supporting 3T3 cells, it must be able to act directly on the keratinocytes. It can also act in the absence of ECF and of medium conditioned by 3T3 cells, although proliferation is greatest when supporting 3T3 cells and EGF are present. The increased proliferation in the presence of the toxin is associated with an increased proportion of small cells known to include the multiplying fraction. The use of toxin makes the cultivation of keratinocytes from epidermis and other stratified squamous epithelia much easier and prolong the culture life of the cells. Whether cell proliferation in the intact epidermis is regulated through agents affecting cAMP (in a direction opposite to that suggested by much of the earlier literature) remains to be elucidated, but the existence of such a mechanism in cultured cells suggests that it may function in the intact epithelium.     

PA‐MSHA inhibits proliferation and induces apoptosis through the up‐regulation and activation of caspases in the human breast cancer cell lines

To investigate the effects of PA‐MSHA (Pseudomonas aeruginosa‐mannose sensitive hemagglutinin) on inhibiting proliferation of breast cancer cell lines and to explore its mechanisms of action in human breast cancer cells. MCF‐10A, MCF‐7, MDA‐MB‐468, and MDA‐MB‐231HM cells were treated with PA‐MSHA or PA (Heat‐killed P. aeruginosa) at different concentrations and different times. Changes of cell super‐microstructure were observed by transmission electron microscopy. Cell cycle distribution and apoptosis induced by PA‐MSHA were measured by flow cytometry (FCM) with PI staining, ANNEXIN V‐FITC staining and Hoechst33258 staining under fluorescence microscopy. Western blot was used to evaluate the expression level of apoptosis‐related molecules. A time‐dependent and concentration‐dependent cytotoxic effect of PA‐MSHA was observed in MDA‐MB‐468 and MDA‐MB‐231HM cells but not in MCF‐10A or MCF‐7 cells. The advent of PA‐MSHA changed cell morphology, that is to say, increases in autophagosomes, and vacuoles in the cytoplasm could also be observed. FCM with PI staining, ANNEXIN V‐FITC and Hoechst33258 staining showed that the different concentrations of PA‐MSHA could all induce the apoptosis and G₀–G₁ cell cycle arrest of breast cancer cells. Cleaved caspase 3, 8, 9, and Fas protein expression levels were strongly associated with an increase in apoptosis of the breast cancer cells. There was a direct relationship with increased concentrations of PA‐MSHA but not of PA. Completely different from PA, PA‐MSHA may impart antiproliferative effects against breast cancer cells by inducing apoptosis mediated by at least a death receptor‐related cell apoptosis signal pathway, and affecting the cell cycle regulation machinery. J. Cell. Biochem. 108: 195–206, 2009. © 2009 Wiley‐Liss, Inc.