Effect of cell cycle on the regulation of the cell surface and secreted forms of type I and type II human tumor necrosis factor receptors

The cell cycle has been shown to regulate the biological effects of human tumor necrosis factor (TNF), but to what extent that regulation is due to the modulation of TNF receptors is not clear. In the present report we investigated the effect of the cell cycle on the expression of surface and soluble TNF receptors in human histiocytic lymphoma U-937. Exposure to hydroxyurea, thymidine, etoposide, bisbensimide, and democolcine lead to accumulation of cells primarily in G₁/S, S, S/G₂/M, G₂/M, and M stages of the cell cycle, respectively. Whilie no significant change in TNF receptors occurred in cells arrested in G₁/S or S/G₂ stages, about a 50% decrease was observed in cells at M phase of the cycle. Scatchard analysis showed a reduction in receptor number rather than affinity. In contrast, cells arrested at S phase (thymidine) showed an 80% increase in receptor number. The decrease in the TNF receptors was not due to changes in cell size or protein synthesis. The increase in receptors, however, correlated with an increase in total protein synthesis (to 3.8-fold of the control levels). A proportional change was observed in the p60 and p80 forms of the TNF receptors. A decrease in the surface receptors in cells arrested in M phase correlated with an increase in the amount of soluble receptors. The cellular response to TNF increased to 8- and 2-fold in cells arrested in G₁ and S phase, respectively; but cells at G2/M phase showed about 6-fold decrease in response. In conclusion, our results demonstrate that the cell cycle plays an important role in regulation of cell-surface and soluble TNF receptors and also in the modulation of cellular response. © 1995 Wiley-Liss, Inc.     

Sensitization to death receptor cytotoxicity by inhibition of fas-associated death domain protein (FADD)/caspase signaling. Requirement of cell cycle progression

Upon binding of their ligands, death receptors belonging to the tumor necrosis factor (TNF) receptor family initiate a signaling pathway leading to the activation of caspases and ultimately apoptosis. TNF, however, in parallel elicits survival signals, protecting many cell types from cell death that can only be induced by combined treatment with TNF and inhibitors of protein synthesis. Here, we report that in NIH3T3 cells, apoptosis in response TNF and cycloheximide is not inhibited by the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD. fmk). Moreover, treatment with zVAD.fmk sensitizes the cells to the cytotoxic action of TNF. Sensitization was also achieved by overexpression of a dominant-negative mutant of Fas-associated death domain protein and, to a lesser extent, by specific inhibition of caspase-8. A similar, but weaker sensitization of zVAD.fmk to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) or anti-CD95 antibody was demonstrated. The unexpected cell death in response to TNF and caspase inhibition occurs despite the activation of nuclear factor kappaB and c-Jun N-terminal kinases. The mode of cell death shows several signs of apoptosis including DNA fragmentation, although activation of caspase-3 was excluded. TNF/zVAD.fmk-induced cell death is preceded by an accumulation of cells in the G(2)/M phase of the cell cycle, indicating an important role of cell cycle progression. This hypothesis is further strengthened by the observation that arresting the cells in the G(1) phase of the cell cycle inhibited TNF/zVAD.fmk-induced cell death, whereas blocking them in the G(2)/M phase augmented it.     

The effect of bleomycin on rapidly proliferating epidermis. A comparative investigation using micro-flow fluorometry, H3Tdr incorporation and a stathmokinetic method (colcemid).

At different time intervals after injection of Bleomycin (BLM) th effect on several kinetic parameters of the hairless mouse epidermis stimulated to proliferate by previous adhesive tape stripping was measured. Micro-flow fluorometry was used to determine the relative number of cells in the various phases of the cell cycle (G1, S and G2). Tritiated thymidine was used to determine labelling indices and grain counts. Colcemid was used to observe the mitotic rate. An initial decrease followed by a subsequent significant increase compared to the non-BLM-treated controls was observed in all parameters studied except the mitotic rate, which remained lower than in the control animals during all 48 hours. The transit time of the cells through the S-phase was initially slightly prolonged, but thereafter it seemed to be shorter than that of the controls. BLM seems to provoke a partial blocking of cells in the G1 phase. When the block is released, a greater number of cells pass through the S phase in partial synchrony at a higher than normal speed. BLM induced a low mitotic rate which remained below the level of that of the normal animals after stripping, even though there obviously was a considerably higher influx of cells from the S phase to the G2 phase. This resulted in a subsequent accumulation of cells in the G2-phase. Thus, BLM has also a blocking effect on the G2-M boundary of the cell cycle. This inhibitory effect of BLM on the mitotic rate was shown to be independent of the effect of BLM on the DNA synthesis. BLM therefore seems to have complex influence on epidermal cell kinetics in vivo. Cells in G1-phase are partially and transiently blocked, but this block is soon released. These cells thereafter pass through the S-phase and pile up in the G2-phase, because BLM also blocks the passage of cells from the G2-phase to mitosis. The overall reduction in cell proliferation seen after BLM in vivo seems mainly to be due to the effect on the G2-M boundray of the cell cycle.     

敲除 Sam68 基因导致白血病细胞系细胞生长迟缓和 G2/M 期延长

RNA 结合蛋白 Sam68 是细胞有丝分裂期 Src 酪氨酸磷酸化的靶蛋白 . 尽管确切机制尚不清楚,一些人还是认为 Sam68 可通过调控 RNA 的代谢参与细胞周期调控 . 利用基因打靶技术,在 DT40 细胞分离出 Sam68 基因缺失的细胞系 . 利用该细胞系,进行 Sam68 的功能解析 . 与野生型细胞系相比, Sam68 基因缺失细胞表现出明显的生长速度迟缓 . 通过细胞周期研究揭示 , 这些细胞生长速度延迟是由于细胞周期中的 G2/M 期延长 . 因为参与细胞周期 G2/M 期调控的周期因子 Cdc2 激酶的活性没有改变,所以提示 Sam68 不依赖于 Cdc2 激酶的活性参与细胞周期中 G2/M 期调控 .     

Effect of recombinant tumor necrosis factor on HL-60 cells: cell-cycle specificity and synergism with actinomycin D

The tumor necrosis factor (TNF) exhibits a multitude of activities depending on the type of target cells. We characterized the cytostatic and cytotoxic effects of recombinant TNF, alone and in combination with actinomycin D (AMD), on the human leukemic cell line HL-60. Because HL-60 cells, when triggered to monocytic differentiation by phorbol esters, are known to produce and secrete TNF, their sensitivity to the factor could indicate an autocrine function of TNF in this cell system. Indeed, HL-60 cells were affected by TNF; their doubling time was increased by about 50% and progression through the cell cycle was perturbed. Initially, (up to 8 h) TNF induced a temporary arrest in G2 while later (24-48 h) it delayed progression through the G1 phase. Also, a transient increase in RNA content peaking at 6-8 h was apparent. The cytotoxicity of TNF alone was low. Thus, TNF may be involved in the regulation of the cell cycle of HL-60 cells during early stages of their differentiation. The cytotoxicity of TNF was markedly potentiated in the presence of AMD; the effect was AMD but not TNF concentration-dependent. Whereas at 20 and 50 ng/ml of AMD alone nonviable cells did not exceed 20% during the first 24 h of treatment, their proportion increased to 80 and 90%, respectively, in the presence of TNF. The most sensitive were cells in the S phase of the cell cycle. The observed synergistic effect of TNF and AMD does not appear to be caused by the action of TNF increasing the permeability of the cell membrane to AMD. The results indicate that HL-60 cells, ordinarily resistant to the cytotoxic action of TNF, can be rendered sensitive by treatment with AMD. This implies that a combination of TNF and AMD may be considered in oncology for treatment of tumors otherwise nonresponding to TNF alone.     

Induction of tumor necrosis factor-alpha and its receptors during differentiation in myeloid leukemic cells along the monocytic pathway. A possible regulatory mechanism for TNF-alpha production

We examined the characteristics of tumor necrosis factor (TNF) receptors expressed on immature mouse myeloid leukemic cells (M1), M1 cells induced to differentiate into macrophages, and macrophage cells (Mm1 cells) by binding studies with radioiodinated TNF. Scatchard analysis of TNF binding revealed that a single class of high affinity receptor was present and that 750-1,100 receptors were expressed on each immature M1 cell. The number of TNF receptors was increased 1.5-2-fold on differentiated M1 cells and 4-5-fold on Mm1 cells with no change in affinity. The addition of interferon-gamma (IFN-gamma) up-regulated the expression of TNF receptors in differentiated M1 cells and Mm1 cells, while immature M1 cells were insensitive to IFN-gamma. The number of TNF receptors on the differentiated cells was increased 4-5-fold by the treatment with IFN-gamma with no change in the binding constant. The affinity of TNF receptors to human TNF-alpha (Kd = 1.7-2.8 nM) was lower than that to murine TNF-alpha (Kd = 0.2-0.7 nM). The assays for cell growth and [3H]thymidine incorporation suggested that no relation exists between the sensitivity of the cells to TNF-alpha and the number of TNF receptors. Enhancement of TNF-mediated cytotoxicity by the treatment with IFN-gamma did not correlate with increases in the number of TNF receptors. Cytolytic assays using L929 cells demonstrated that the amount of constitutive and lipopolysaccharide (LPS)-induced secretion of TNF-alpha was markedly increased during differentiation. Both the constitutive expression and IFN-gamma-mediated superinduction of TNF receptors, and the constitutive and LPS-induced secretion of TNF-alpha were closely related to the extent of cellular differentiation along the monocytic pathway. The time course of LPS-induced TNF-alpha activity showed a rise-and-decline profile with a peak at 2 h. On the other hand, the time course of the number of cell surface TNF receptors showed a decline-and-rise profile, a mirror image of the TNF-alpha activity time course profile in the supernatant. Anti-TNF-alpha antibody treatment blocked the LPS-induced down-regulation of TNF receptors and increased TNF-alpha mRNA accumulation. We discussed "an autoinhibitory system" in which an internalization of secreted TNF-alpha mediated by its own receptors is involved not only in decreasing TNF-alpha activity in the supernatant but also in reducing TNF-alpha mRNA expression.     

Down regulation of the receptors for tumor necrosis factor by interleukin 1 and 4 beta-phorbol-12-myristate-13-acetate

Binding of radiolabeled tumor necrosis factor (TNF) to cell surface receptors was markedly reduced in human foreskin fibroblasts and cells from SV-80 and HeLa cell lines subsequent to treatment with interleukin 1 (IL-1) or 4 beta-phorbol-12-myristate-13-acetate (PMA). The decrease in TNF binding was initiated within minutes of application of IL-1 or PMA and could not be blocked by cycloheximide, suggesting that it is independent of protein synthesis. Scatchard plot analysis of TNF binding to the SV-80 cells indicated that its decrease in response to IL-1 and PMA reflects a reduced amount of TNF receptors, with no change in their affinity. IL-1 and PMA together had an additive effect on TNF binding. Treatment with TNF did not result in decreased binding of IL-1 to its receptors nor did TNF and IL-1 compete directly for their respective receptors. Human U937 cells on which receptors for IL-1 were below detectable levels exhibited no decrease in TNF binding when treated with IL-1, but did so in response to PMA. In addition to a decrease in TNF receptors, cells treated with IL-1 or PMA exhibited a lesser vulnerability to the cytolytic effect of TNF. The two kinds of changes were not completely correlated. A particularly notable dissimilarity was evident when comparing the rate of their reversal: the TNF receptor level was fully recovered within a few hours of removal of IL-1 or of the water-soluble analogue of PMA, 4 beta-phorbol-12,13-dibutyrate, from pretreated SV-80 cells; yet at that time resistance to the cytotoxicity of TNF was still prominent. These findings indicate that IL-1 as well as tumor-promoting phorbol diesters can down regulate cellular response to TNF by inducing a decrease in the number of receptors for TNF, and apparently through some other effect(s) as well.     

Macrophage cell cycling: influence on Fc receptors and antibody-dependent phagocytosis

The influence of cell cycling on the density and binding properties of IgG2a Fc receptors and their associated antibody-dependent phagocytic activity was investigated with the P388D1 murine macrophage cell line. Unseparated macrophages and subpopulations of elutriated macrophages, enriched for cells in G1, S, and G2 + M phases were compared to detect possible differences in IgG2a-dependent phagocytosis. Suspensions of G2 + M phase cells were appreciably enhanced in phagocytic activity over G1-phase cells, which were less phagocytic than unseparated macrophage populations. An analysis of the binding of 125I-IgG2a myeloma protein disclosed that the IgG2a Fc receptor avidity remained essentially unchanged during cell cycle traverse, whereas the number of IgG2a Fc receptors more than doubled as cells cycled from G1 to G2 + M (1.5 X 10(5) vs 3.4 X 10(5) receptors per cell). With their increased size relative to G1 cells, and the resultant increase in receptor number, G2 phase cells should have more productive collisions with the antibody-coated target cells and greater phagocytic capacity.     

Species specificity of human and murine tumor necrosis factor. A comparative study of tumor necrosis factor receptors

Recombinant murine and human tumor necrosis factor (mTNF and hTNF, respectively) were radioiodinated to high specific activity using a solid-phase lactoperoxidase method. A single class of high affinity receptors for 125I-TNF was identified on TNF-sensitive murine L cells and human HeLa S2 cells. Competitive radioligand binding assays were used to study the species specificity of TNF preparations. Unlabeled hTNF competed 30-fold less effectively than mTNF for binding to L cell receptors, whereas mTNF competed to approximately the same extent as hTNF for binding to HeLa cell receptors. A similar species specificity was observed in cytotoxicity assays; hTNF was more cytotoxic for HeLa cells than mTNF. Conversely, mTNF was more growth inhibitory and cytotoxic for L cells than hTNF. mTNF. and hTNF.receptor complexes were compared by gel filtration chromatography and polyacrylamide gel electrophoresis before and after cross-linking with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES). These complexes eluted in gel filtration at a position corresponding to a globular protein of 350,000 Mr. Gel autoradiographs of the fractions containing cross-linked complexes showed bands of 95,000 and 75,000 Mr as well as small amounts of higher Mr bands. mTNF and hTNF treated with BSOCOES formed cross-linked dimers and trimers. Therefore, we were unable to determine whether the 95,000 and 75,000 Mr bands represented two distinct subunits of receptors or one subunit to which either a dimer or a monomer of TNF was cross-linked. These results demonstrate species specificity in the TNF-receptor interaction. In addition, the affinity labeling studies in two species give an identical pattern for the TNF X receptor complexes, suggesting that the receptors have similar subunit composition.     

Interferon-gamma enhances expression of cellular receptors for tumor necrosis factor

Incubation of several human tumor cell lines with human interferon-gamma (IFN-gamma) increased the specific binding of subsequently added 125I-labeled recombinant human tumor necrosis factor (TNF). A similar increase in TNF binding was seen in murine L929 cells after incubation with murine IFN-gamma, but not after incubation with human IFN-gamma. Increased TNF binding to cells incubated with IFN-gamma was due to an increase in the number of TNF receptors, with no demonstrable change in binding affinity. In one out of two human cell lines tested, IFN-alpha and IFN-beta also produced increased TNF binding, albeit with a lower efficacy than IFN-gamma. A maximal increase in TNF binding was seen after about 6 to 12 hr of incubation with IFN. Increased TNF binding due to enhanced TNF receptor expression may contribute to the enhancement of TNF cytotoxicity seen in some tumor cell lines after INF treatment. Modulation of TNF receptor expression by IFN may also influence other biological activities of TNF.     

The active form of tumor necrosis factor is a trimer

Natural human and recombinant human and murine tumor necrosis factors (TNF) were fractionated by gel filtration chromatography on Sephadex G-75. The active form of TNF was identified by its inhibitory activity in receptor binding assays with HeLa cells and was eluted as a protein of Mr approximately 55,000. Radioiodinated human and murine TNF were fractionated by gel filtration into a major peak of Mr approximately 55,000, corresponding to a trimer, and a minor peak of Mr approximately 17,000, corresponding to a monomer. Binding assays showed that the timer was at least 8-fold more active than the monomer. The human TNF partially dissociated into monomers upon addition of the nonionic detergent Triton X-100. Isolated monomers showed low binding affinity (KD = 70 nM) and reduced cytotoxicity, whereas trimers showed high binding affinity (KD = 90 pM) and cytotoxicity. When 125I-TNF was bound to cells, no release of monomer was detectable, suggesting that the trimer could directly bind to cellular receptors without dissociating into subunits. Further evidence for such binding was obtained by cross-linking 125I-TNF trimers with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone. These trimers were bound to HeLa cells, could be dissociated from cellular receptors, and elicited a cytotoxic response. These results show that trimers, whether native or cross-linked, bind to receptors and are the biologically active form of TNF.     

Fluctuation of trypsin-like proteinase activity in the cell cycle of synchronized and growing HeLa cells, and the effect of GMCHA-OPhBut

HeLa cells synchronized by double-thymidine block were grown in Eagle's minimum essential medium supplemented with 10% calf serum, and the fluctuation of trypsin-like protease activity in the cell cycle was examined. Seven distinct activity peaks were observed in one cell cycle at a cell density of 2%: two peaks in S phase, one peak at the S/G2 boundary, one peak in early M phase and one at the M/G1 boundary, and two peaks in G1 phase. HeLa cells synchronized by a mitotic detachment technique also showed similar results at cell density of 4.8%. The appearance of trypsin-like proteinase activity in the cell cycle was markedly affected by cell density, and no definite peak was observed above 8%. trans-Guanidinomethylcyclohexanecarboxylic and 4-tert-butylphenyl ester (GMCHA-OPhBut), a specific inhibitor for trypsin and a strong inhibitor of HeLa cell growth, had no effect on the various events in the first S, G2 and M phases, such as the incorporation of [methyl-3H]thymidine into DNA, the increase in the cell concentration, and the appearance of trypsin-like proteinase activity, whereas it retarded the onset of the second S phase and the various events in the second S, G2 and M phases for 3 h. In particular, it induced the appearance of a new proteinase peak at the G1/S boundary.     

Synthesis of poly(adenosine diphosphate ribose) in synchronized Chinese hamster cells.

Chinese hamster ovary cells were synchronized by mitotic selection and used to study the relation of poly(adenosine diphosphate ribose) synthesis to DNA synthesis and the different phases of the cell cycle. DNA synthesis was measured in cells rendered permeable to exogenously supplied nucleotides. Poly(ADPR) synthesis was also measured in permeable cells in the presence of both minimum and maximum DNA damage. The maximum DNA damage was produced by treating the cells with saturating concentrations of DNase. As anticipated, the DNA synthesis complex showed its maximum activity during S phase and showed 4–5-fold less activity during the other phases of the cell cycle. The basal level of poly(ADPR) synthesis was elevated during G1, fell to its lowest level during S phase, then increased during G2 and rose to its highest level during G1. The DNase responsive activity of poly(ADPR) synthesis was relatively constant thru the cell cycle but showed a peak at the end of S phase; then the activity decreased during the subsequent G2-M period.     

Inhibitors of ADP-ribose polymerase decrease the resistance of HER2/neu-expressing cancer cells to the cytotoxic effects of tumor necrosis factor.

Four human ovarian and breast tumor lines expressing the HER2/neu oncogene were resistant to the cytotoxic and DNA-degradative activity of TNF. The resistance was not associated with altered TNF receptor function because Scatchard analysis of 125I-rTNF binding to HER2/neu-expressing target cells revealed receptors with normal binding parameters. Furthermore, the TNF receptors on the resistant lines were capable of signal transduction as evidence by the induction of ADP-ribose polymerase activity and MHC expression. TNF resistance was not reversed by coincubation with drugs that interrupted the glutathione redox cycle. In addition, although coincubation of HER2/neu-expressing targets with cycloheximide resulted in significant TNF-induced lysis, when compared to HER2/neu-nonexpressing targets similarly treated with cycloheximide, a significant relative resistance was still present. To investigate the role of ADP-ribosylation in the resistance of these targets, we used nontoxic concentrations of two inhibitors of ADP-ribose polymerase, 3-aminobenzamide, and nicotinamide. Both inhibitors completely reversed the resistance of HER2/neu-expressing targets to TNF-mediated cytotoxicity and DNA injury in a concentration-dependent fashion. These inhibitors of ADP-ribose polymerase did not act by down-regulating expression of HER2/neu oncogenes. In contrast, aminobenzamide and nicotinamide significantly diminished TNF-induced cytotoxicity of L929 targets. These data suggest that the activity of ADP-ribose polymerase may play a pivotal role in determining the fate of the target cell during exposure to TNF.     

The cell cycle phases of DNA damage and repair initiated by topoisomerase II-targeting chemotherapeutic drugs

Although cytostasis and cytotoxicity induced by cancer chemotherapy drugs targeting topoisomerase II (topoII) arise in specific cell cycle phases, it is unknown whether the drug-initiated DNA damage triggering these responses, or the repair (reversal) of this damage, differs between cell cycle phases or between drug classes. Accordingly, we used a flow cytometric alkaline unwinding assay to measure DNA damage (strand breakage (SB)) and SB repair in each cell cycle compartment of human cancer cell lines treated with clinically relevant concentrations of doxorubicin, daunomycin, etoposide, and mitoxantrone. We found that treated HeLa and A549 cells exhibited the greatest SB in G2/M phase, the least in G1 phase, and generally an intermediate amount in S phase. The cell cycle phase specificity of the DNA damage appeared to be predictive of the cell cycle phase of growth arrest. Furthermore, it appeared to be dependent on topoIIalpha expression as the extent of SB did not differ between cell cycle compartments in topoIIalpha-diminished A549(VP)28 cells. HeLa cells were apparently unable to repair doxorubicin-initiated SB. The rate of repair of etoposide-initiated SB in HeLa cells and of mitoxantrone-initiated SB in HeLa and A549 cells was similar in each cell cycle compartment. In A549 cells, the rate of repair of doxorubicin and etoposide-initiated SB differed between cell cycle phases. Overall, these results indicate that the cell cycle phase specificity of cytostasis and cytotoxicity induced in tumor cells by topoII-targeting drugs may be directly related to the cell cycle phase specificity of the drug-initiated DNA damage. Analysis by cell cycle compartment appears to clarify some of the intercellular heterogeneity in the extent of drug-initiated DNA damage and cytotoxicity previously observed in cancer cells analyzed as a single population; this approach might be useful in resolving inconsistent results reported in investigations of tumor cell topoII content versus response to topoII-targeting drugs.     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.     

A new proteasome inhibitor, TP-110, induces apoptosis in human prostate cancer PC-3 cells

Proteasome inhibitors are useful in the treatment of cancer. Recently, we found a new proteasome inhibitor, TP-110, derived from tyropeptin A produced by Kitasatospora sp. Here we report that TP-110 induces apoptosis in human prostate cancer PC-3 cells. TP-110 showed strong cytotoxicity to PC-3 cells (IC(50)=0.05 muM). It increased the number of cells in the G(2)-M phase and increased the accumulated amounts of the p21 and p27 proteins, which are negative regulators of cell cycle progression. Furthermore, it induced apoptosis along with chromatin condensation and DNA fragmentation in PC-3 cells, and TP-110-induced apoptosis appeared to be associated with caspase activation. Additionally, TP-110 inhibited not only the degradation of IkappaB and the nuclear translocation of nuclear factor-kappaB (NF-kappaB), but also the DNA binding activity of NF-kappaB. These results indicate that TP-110 shows a strong growth inhibition and apoptosis in PC-3 cells.     

Binding of 14-3-3beta to the carboxyl terminus of Wee1 increases Wee1 stability, kinase activity, and G2-M cell population.

Wee1 protein kinase plays an important regulatory role in cell cycle progression. It inhibits Cdc-2 activity by phosphorylating Tyr15 and arrests cells at G2-M phase. In an attempt to understand Wee1 regulation during cell cycle, yeast two-hybrid screening was used to identify Wee1-binding protein(s). Five of the eight positive clones identified encode 14-3-3beta. In vivo binding assay in 293 cells showed that both full-length and NH2-terminal truncated Wee1 bind with 14-3-3beta. The 14-3-3beta binding site was mapped to a COOH-terminal consensus motif, RSVSLT (codons 639 to 646). Binding with 14-3-3beta increases the protein level of full-length Wee1 but not of the truncated Wee1. Accompanying the protein level increases, the kinase activity of Wee1 also increases when coexpressed with 14-3-3beta. Increased Wee1 protein level/enzymatic activity is accountable, at least in part, to an increased Wee1 protein half-life when coexpressed with 14-3-3beta. The protein half-life of the NH2-terminal truncated Wee1 is much longer than that of the full-length protein and is not affected by 14-3-3beta cotransfection. Biologically, 14-3-3beta/Wee1 coexpression increases the cell population at G2-M phase. Thus, Wee1 binding with 14-3-3beta increases its biochemical activity as well as its biological function. The finding reveals a novel mechanism by which 14-3-3 regulates G2-M arrest and suggests that the NH2-terminal domain of Wee1 contains a negative regulatory sequence that determines Wee1 stability.