[Remote results of semiclosed endarterectomy with a loop from the superficial femoral artery and femoropopliteal bypass grafting].

AIM: To compare the results of semiclosed retrograde loop endarterectomy from the superficial femoral artery (SFA) and proximal femoropopliteal bypass grafting (FPBG) with a synthetic prosthesis. MATERIAL AND METHODS: From 2007 to 2011, at the Department of Vascular Surgery of Municipal Clinical Hospital No 15 named after O.M. Filatov we operated on a total of 97 patients with occlusive and stenotic lesions of the femoropopliteal segment, with 83 (86%) of them being men. The mean age of the patients was 62.7 ± 8.9 years. By the degree of chronic ischaemia the patients were subdivided according to the Fontain-Pokrovsky classification as follows: 52 (53.6%) patients had stage 2B ischaemia, 22 (22.7%) patients presented with stage 3, and 14 (14.4%) patients had stage 4. Nine (9.3%) patients were diagnosed with symptoms of acute ischaemia. Depending on the operative treatment performed, the patients were subdivided into two groups: Group I (n=56) comprising patients subjected to FPBG above the knee-joint fissure and Group II (n=41) consisting of those who underwent loop endarterectomy from the SFA. RESULTS: Technical success was obtained in 97 (100%) patients. After the operation the average ankle-brachial index (ABI) amounted to 0.87 ± 0.22 in Group I and 0.83 ± 0.22 in Group II. One (1.8%) patient from Group I developed shunt thrombosis successfully treated by thrombectomy. In Group II after surgery we also observed 1 thrombosis (2.4%) of the desobliterated superficial femoral artery, treated by thrombectomy from the SFA. The remote results were followed up during 4-7 years. Primary patency in the group of patients undergoing FPBG amounted after 1 and 2 years to 73.1% and 63.2% (p<0.005), and after 3 and 4 years to 34.1% and 30.7% (p>0.05). In the group of patients after loop EAE from the SFA 1-, 2-, 3- and 4-year primary patency amounted to 92.1%, 74.6% (p<0.05) and 63.1%, 58.9% (p>0.05), respectively. CONCLUSION: Semiclosed retrograde loop endarterectomy (EAE) demonstrated better results both in the immediate and remote postoperative periods as compared with femoropopliteal proximal bypass grafting using a synthetic prosthesis.

[Hybrid operations in multilevel lesions of lower-limb arteries].

Reflected in the article is experience in treating a total of 24 patients suffering from lower limb chronic ischaemia and diagnosed as having concomitant lesions of the aortofemoral and femoropopliteal segments, with a total of 24 hybrid operations performed. Of these, nineteen interventions consisted of implanting a stent graft into iliac arteries and revascularization of the femoropopliteal segment, whereas the remaining five cases included balloon angioplasty of the iliac artery with revascularization of the femoropopliteal segment. Good immediate outcomes were obtained in 23 patients. The ankle-brachial index increased averagely from 0.38 to 0.88. The remote outcomes (from 12 months to 5 years) were followed up in fifteen patients. Cumulative patency of the bypass grafts after 1, 3 and 5 years amounted to 78%, 41.5% and 41.5%, respectively. The cumulative survival rates after 1, 3 and 5 years equalled 90.8%, 76.2% and 76.2%, respectively. All extremities were eventually spared, with the long-term limb salvage rate thus amounting to 100%.

Study of the cytolethal distending toxin (CDT)-activated cell cycle checkpoint. Involvement of the CHK2 kinase.

The bacterial cytolethal distending toxin (CDT) triggers a G2/M cell cycle arrest in eukaryotic cells by inhibiting the CDC25C phosphatase-dependent CDK1 dephosphorylation and activation. We report that upon CDT treatment CDC25C is fully sequestered in the cytoplasmic compartment, an effect that is reminiscent of DNA damage-dependent checkpoint activation. We show that the checkpoint kinase CHK2, an upstream regulator of CDC25C, is phosphorylated and activated after CDT treatment. In contrast to what is observed with other DNA damaging agents, we demonstrate that the activation of CHK2 can only take place during S-phase. Use of wortmannin and caffeine suggests that this effect is not dependent on ATM but rather on another as yet unidentified PI3 kinase family member. These results confirm that the CDT is therefore responsible for specific genomic injuries that block cell proliferation by activating a cell cycle checkpoint.

Study of the cytolethal distending toxin-induced cell cycle arrest in HeLa cells: involvement of the CDC25 phosphatase.

HeLa cells exposed to Escherichia coli cytolethal distending toxins (CDT) arrest their cell cycle at the G2/M transition. We have shown previously that in these cells the CDK1/cyclin B complex is inactive and can be reactivated in vitro using recombinant CDC25 phosphatase. Here we have investigated in vivo the effects of CDC25 on this cell cycle checkpoint. We report that overexpression of CDC25B or CDC25C overrides an established CDT-induced G2 cell cycle arrest and leads the cells to accumulate in an abnormal mitotic stage with condensed chromatin and high CDK1 activity. This effect can be counteracted by coexpression of the WEE1 kinase. In contrast, overexpression of CDC25B or C prior to CDT treatment prevents G2 arrest and allows most of the cells to progress through mitosis with only a low percentage of cells arrested in abnormal mitosis. The implications of these results on the biochemical nature of the CDT-induced cell cycle arrest are discussed.

[Role of CDC25 phosphatases in the control of proliferation]. / Rôle des phosphatases CDC25 dans le contrôle de la prolifération.

Progression in cell cycle is controlled by CDKs (cyclin dependent kinases) and their cyclins regulatory subunits. In mammalian cells, three dual specificity phosphatases called CDC25 activate CDKs/cyclin complexes. The activity of CDC25 is regulated by phosphorylation and dephosphorylation events. CDC25 phosphatases also participate in cell cycle checkpoints activated in response to DNA damage. Two members of this family, CDC25 A and CDC25 B, have oncogenic properties, and their overexpression has been detected in various types of tumors.

Regulation of CDC25B phosphatases subcellular localization.

The CDC25B dual specificity phosphatase is involved in the control of the G2/M transition of the cell cycle. Subcellular localization might represent an important aspect of the regulation of its activity. We have examined in transiently transfected asynchronous HeLa cells the localization of HA-tagged CDC25B proteins and found that they are nuclear or cytoplasmic suggesting the existence of an active shuttling. Accordingly, localization analysis of deletion and truncation proteins indicates that CDC25B contains a putative nuclear localization signal located between residues 335 and 354. We also demonstrated that a short 58 residues deletion of the amino-terminus end of CDC25B is sufficient to retain it to the nucleus. Mutational analysis indicates that a nuclear export sequence is located between residues 28 and 40. In addition, treatment of the cells with the exportin inhibitor, Leptomycin B, has the same effect. The mutation of Ser-323, a residue that is essential for the interaction with 14-3-3 proteins, also abolishes cytoplasmic staining. The subcellular localization of CDC25B is therefore dependent on the combined effects of a nuclear localization signal, a nuclear export signal and on the interaction with 14-3-3 proteins.

14-3-3 proteins and growth control.

The 14-3-3 proteins constitute a family that is highly conserved in a wide range of organisms, including higher eukaryotes, invertebrates and plants. Variants of 14-3-3 proteins assembled in homo- and heterodimers were found to interact with diverse cellular proteins. Until recently, the biological role of 14-3-3 members was still poorly understood. However, the results of an increasing number of studies on their structure and function are converging to define 14-3-3 proteins as a novel type of adaptor that modulates interactions between components involved in signal transduction pathway and in cell cycle control.

Specific interaction between 14-3-3 isoforms and the human CDC25B phosphatase.

CDC25 dual-specificity phosphatases are essential regulators that activate cyclin-dependent kinases (CDKs) at critical stages of the cell cycle. In human cells, CDC25A and C are involved in the control of G1/S and G2/M respectively, whereas CDC25B is proposed to act both in S phase and G2/M. Evidence for an interaction between CDC25 phosphatases and members of the 14-3-3 protein family has been obtained in vitro and in vivo in several organisms. On the basis of the work performed with CDC25C, it has been proposed that phosphorylation is required to mediate the interaction with 14-3-3. Here we have examined the molecular basis of the interaction between CDC25B phosphatases and 14-3-3 proteins. We show that in the two-hybrid assay all three splice variants of CDC25B interact similarly and strongly with 14-3-3eta, beta and zeta proteins, but poorly with epsilon and Theta. In vitro, CDC25B interacts at a low level with 14-3-3beta, epsilon, zeta, eta, and Theta isoforms. This interaction is not increased upon phosphorylation of CDC25B by CHK1 and is not abolished by dephosphorylation. In contrast, a specific, strong interaction between CDC25B and 14-3-3zeta and eta isoforms is revealed by a deletion of 288 residues in the amino-terminal region of CDC25B. This interaction requires the integrity of Ser 323, although it is independent of phosphorylation. Thus, interaction between 14-3-3 proteins and CDC25B is regulated in a manner that is different from that with CDC25C. We propose that, in addition to a low affinity binding site that is available for all 14-3-3 isoforms, post-translational modification of CDC25B in vivo exposes a high-affinity binding site that is specific for the zeta and eta14-3-3 isoforms.

Phosphorylation and proteasome-dependent degradation of Bcl-2 in mitotic-arrested cells after microtubule damage.

Treatment of NIH-OVCAR-3 cells with paclitaxel, a microtubule-stabilizing agent, induces mitotic arrest and apoptosis, but also Bcl-2 phosphorylation. We report here that Bcl-2 phosphorylation precedes Bcl-2 down-regulation and that both events are closely associated with mitotic arrest, but are not sufficient for paclitaxel to trigger apoptosis. Indeed, when paclitaxel-treated cells were induced to exit mitosis in the presence of 2-aminopurine, Bcl-2 phosphorylation and Bcl-2 down-regulation were both inhibited. In contrast, when apoptosis was inhibited by a caspase inhibitor or Bcl-2 over-expression, Bcl-2 phosphorylation and down-regulation still occurred. Furthermore, we show that Bcl-2 is degraded in mitosis by the proteasome-dependent pathway since Bcl-2 down-regulation is inhibited by proteasome inhibitors such as MG132, Lactacystin and LLnL. Taken together these results indicate that mitotic spindle damage results in post-translational modifications of Bcl-2 by phosphorylation and degradation.

Use of CDC2 from etoposide-treated cells as substrate to assay CDC25 phosphatase activity.

Cyclin-dependent kinases (CDKs) regulate the key transition of the cell cycle in all organisms. In response to Etoposide (VP-16) induced DNA damage, cells undergo a G2-phase arrest resulting in the accumulation of inactive CDK1 (CDC2) kinase complexes. Here we report that upon Etoposide treatment CDC2 is phosphorylated on tyrosine 15 and is dephosphorylated and activated in vitro by recombinant CDC25 phosphatase. We also show that inactive CDC2 kinase from Etoposide-treated cells can be used as a substrate in a sensitive two-step assay of CDC25 phosphatase. This assay, which is very simple to set-up, is based on the monitoring of CDC2 kinase activity after CDC25-dependent dephosphorylation. It provides the possibility to use a highly physiological substrate in antimitotic drugs screening.

Proteasome-dependent degradation of human CDC25B phosphatase.

The CDC25 dual specificity phosphatase is a universal cell cycle regulator. The evolutionary conservation of this enzyme from yeast to man bears witness to its major role in the control of cyclin-dependent kinases (CDK) activity that are central regulators of the cell cycle machinery. CDC25 phosphatase both dephosphorylates and activates CDKs. Three human CDC25s have been identified. CDC25A is involved in the control of G1/S, and CDC25C at G2/M throught the activation of CDK 1-cyclin B. The exact function of CDC25B however remains elusive. We have found that CDC25B is degraded by the proteasome pathway in vitro and in vivo. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex, but not by CDK1-cyclin B. Together with the observations of others made in yeast and mammals, our results suggest that CDC25B might act as a 'mitotic starter' triggering the activation of an auto-amplification loop before being degraded.

Increased level of p21 in human ovarian tumors is associated with increased expression of cdk2, cyclin A and PCNA.

We have demonstrated over-expression of the cyclin-dependent kinase inhibitor p21 in various ovarian-cancer cell lines as well as in ovarian-tumor biopsies. This increase in p21 expression relative to that observed in normal ovarian epithelial cells is unrelated to proliferation index. In the present study, we found that p21 is functional, since the protein extracted from IGROVI cells is still able to inhibit cdk2-kinase activity. We then investigated how IGROVI cells overcome the growth-inhibitory function of p21. Immunofluorescence assays and subcellular fractionation showed that p21 is located in cytoplasm and nucleus both in normal and in tumoral cells. Compared with normal ovarian epithelial cells in culture, the increase in level of p21 in IGROVI cells was found to be associated with increased expression of cdk2, cyclin-A and PCNA proteins. In IGROVI cells, p21 is associated with inactive cdk2/cyclin-A complex, indicating that it acts as an inhibitory factor rather than an assembly factor. Over-expression of cdk2 and of cyclin A observed in IGROVI cells allows them to escape to p21-inhibitory activity. The fact that cells from ovarian-tumor biopsies exhibited a concomitant increase in p21 and in its partners cdk2 and PCNA suggest that ovarian-tumor cells can tolerate high levels of functional p21 via over-expression of other cell-cycle-regulatory proteins.

Evidence for a mammalian Nim1-like kinase pathway acting at the G0-1/S transition.

In fission yeast the Nim1 kinase phosphorylates and inactivates the cdc2-inhibitory Weel tyrosine kinase. In addition, Nim1 is necessary for an efficient cellular response to nutritional starvation leading to cell cycle arrest in G1. Given the remarkable evolutionary conservation of the cell cycle regulatory mechanism we have investigated the effect of Nim1 expression on the control of the mammalian cell cycle using a plasmid microinjection approach. In synchronised IMR90 human fibroblasts, expression of Nim1 strongly inhibited entry into S-phase. This effect was dependent on the catalytic activity of Nim1 and did not require its regulatory domain. Furthermore we show that co-expression of human Wee1 kinase reverted the inhibitory effect, indicating that Nim1 was acting in a Wee1-dependent manner. These results provide evidence for the existence of a Nim1-like kinase pathway acting at the G0-1/S transition in human cells.

Alternative splicing of the human CDC25B tyrosine phosphatase. Possible implications for growth control?

CDC25B2, a protein tyrosine phosphatase closely related to the putative CDC25B oncogene, was identified in a Burkitt lymphoma cDNA library. CDC25B2 differs from CDC25B by a 14 residue insertion and a 41 residue deletion, which are both located in the amino-terminal region of the protein, upstream of the catalytic domain. Examination of the genomic sequence revealed that CDC25B1 (formerly B) and CDC25B2 are splice variants of the same gene. A third variant, CDC25B3, that carries both the 14 and the 41 residue sequences was also identified in the same cDNA library. All three variants were detected in a panel of human primary culture and cell lines, although at different levels. In primary fibroblasts and in HeLa cells the CDC25B expression is cell cycle regulated, reaching a maximum in G2-phase. In vitro, CDC25B1 phosphatase is slightly more active than CDC25B2 and B3. However, episomal overexpression of the three CDC25B variants in fission yeast reveals that in vivo, CDC25B2 is largely more active than either B1 or B3 (B2>B3>B1) both to complement a thermosensitive S pombe CDC25 activity and to act as a mitotic inducer. Alternative splicing of CDC25B may therefore contribute to the control of cell proliferation.

Cyclin D1 expression is a major target of the cAMP-induced inhibition of cell cycle entry in fibroblasts.

We previously described in the CCL39 hamster fibroblast cell line the inhibition of DNA synthesis reinitiation by agents that elevate cyclic AMP. Here, we show that 8Br-cAMP strongly blocks both the growth factor-induced increase in cyclin D1 protein expression and decrease in p27(KIP1) protein levels, leaving untouched the levels of cyclin D3, cdk2 and cdk4. To assess the role of cyclin D1 in the cAMP-mediated inhibition of DNA synthesis, we overexpressed the cyclin D1 gene in CCL39 and analysed the cAMP response in stable transfectants. We showed that the kinase activities associated to G1 cyclin-cdk complexes are significantly more resistant to cAMP in cyclin D1 transfectants than in their normal counterparts, although the serum-induced p27(KIP1) disparition is still cAMP sensitive in cyclin D1 overexpressors. Interestingly, the mitogen-induced DNA synthesis reinitiation is also much less inhibited by cAMP in cyclin D1 transfectants than in control cells. These data clearly establish that the cAMP-inducible blockade of the G1 phase of the cell cycle can be partially alleviated by overexpression of cyclin D1 in hamster fibroblasts, thus strongly suggesting that cyclin D1 protein is one of the major targets for cAMP inhibitory action in fibroblasts.

Phosphorylation of human CDC25B phosphatase by CDK1-cyclin A triggers its proteasome-dependent degradation.

In eukaryotes the activity of CDK1 (CDC2), a cyclin-dependent kinase that initiates the structural changes that culminate in the segregation of chromosomes at mitosis, is regulated by the synergistic and opposing activities of a cascade of kinases and phosphatases. Dephosphorylation of threonine 14 and tyrosine 15 of CDK1 by the CDC25 phosphatases is a key step in the activation of the CDK1-cyclin B protein kinase. Little is currently known about the role and the regulation of CDC25B. Here we report in vitro and in vivo data that indicate that CDC25B is degraded by the proteasome. This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex but not by CDK1-cyclin B. These results indicate that CDK1-cyclin A phosphorylation targets CDC25B for degradation and that this might be an important component of cell cycle regulation at the G2/M transition.

Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line.

It has been shown recently that expression of p21 is enhanced by paclitaxel. This cytotoxic compound induces mitotic spindle damage resulting in blockade of the mitotic cell cycle associated or not with apoptotic cell death. In the present study, we showed that, in MCF-7 cells, paclitaxel induced accumulation of p21 in cells with a G2/M DNA content, corresponding to cells either in abnormal mitosis or in an interphase-like state (decondensed chromatin) with multiple nuclei. In MCF-7 cells, the increase in p21 was subsequent to the mitotic arrest and was associated with the exit from abnormal mitosis leading to formation of cells with micronuclei. In this cell line, we noted a relationship between the elevation of p21 expression and the inhibition of p34cdc2 activity. High levels of p21 protein were also found to be associated with inactive p34cdc2/cyclin B protein complex after treatment with paclitaxel. Treatment with p21 antisense oligonucleotide partially blocked induction of p21 expression by paclitaxel and significantly reduced survival of MCF-7 cells exposed to this agent. In NIH-OVCAR-3 cells, which are deficient in basal and paclitaxel-induced p21 expression, paclitaxel led to a prolonged activation of p34cdc2 and a delayed mitotic exit associated with apoptotic cell death. These observations suggest that p21 is not required for the mitotic arrest in response to paclitaxel, but argue in favor of a role for this inhibitor in facilitating the exit from abnormal mitosis. This effectively enhances cell survival after paclitaxel-induced spindle damage.

Expression of p21WAF1/CIP1 is heterogeneous and unrelated to proliferation index in human ovarian carcinoma.

The growth-inhibitory protein p21WAF1/CIP1 is a potent inhibitor of various cyclin-dependent kinases, the expression of which is regulated at the transcriptional level by p53-dependent and -independent mechanisms. We examined p21WAF1/CIP1 mRNA and protein expression in 5 human ovarian-adenocarcinoma cell lines, 1 primary culture of normal surface epithelium and 17 human ovarian-tumor specimens. In culture cells, the p21WAF1/CIP1 protein was expressed in normal ovarian epithelial cells and at a high level in the adenocarcinoma 2008 and IGROV-1 cell lines. p21 WAF1/CIP1 expression was undetectable at the mRNA and protein levels in the NIH-OVCAR-3 and SKOV-3 ovarian-adenocarcinoma cell lines which are respectively mutated and deleted in the p53 gene. Heterogeneous expression of p21WAF1/CIP1 observed in ovarian-cancer cell lines in culture was also found in vivo on tumor specimens. p21WAF1/CIP1 expression is undetectable in 25% of the ovarian biopsies examined. Since it has been found that the p53 gene is mutated in 79% of ovarian cancer, the absence of p21WAF1/CIP1 expression in 25% of these ovarian cancer could not be correlated with p53 mutation. The proliferation index of the 17 tumors showed great variation from one tumor to another. However, no significant correlation was found between p21WAF1/CIP1 expression and the proliferation rate of the tumors.

Subcellular localisation of human wee1 kinase is regulated during the cell cycle.

Wee1 kinase-dependent phosphorylation of cdc2 maintains the cdc2/cyclin B complex in an inert form until it is activated by the cdc25 tyrosine phosphatase at the end of G2. As described for cdc25, cell cycle-linked changes in the intracellular localisation of wee1 may constitute an important aspect of the temporal regulation of cdc2 activity. Here we report that the subcellular distribution of human wee1 changes during the cell cycle in HeLa and IMR90 cells. During interphase, wee1 is found almost exclusively in the nucleus. When the cell enters mitosis, wee1 is relocalised into the cytoplasm. During cell division, wee1 becomes restricted to the mitotic equator and by the end of mitosis it is found exclusively in association with midbody bridges, a phenomenon that is dependent on microtubule assembly. The relocalisations of wee1 and its association with subcellular structures may play key regulatory roles at different stages of the cell cycle and during mitosis.

Effects of TGF-beta 1 (transforming growth factor-beta 1) on the cell cycle regulation of human breast adenocarcinoma (MCF-7) cells.

The antiproliferative effects of TGF-beta 1 were investigated in a human breast adenocarcinoma cell line (MCF-7). We report that TGF-beta 1 inhibits proliferation through cell cycle arrest in G1. A MCF-7 cell subline (MCF-7(-)), in which the type II TGF-beta receptor is not detected, was shown to be resistant to TGF-beta 1 growth inhibitory effect. Cdk2 kinase activity was inhibited in the MCF-7 sensitive cell subline in parallel with the inhibition of cell cycle progression. In both sensitive and resistant cell lines, TGF-beta 1 treatment did not affect cdk2, cdk4, cyclin E and cyclin D1 mRNA and protein levels. However, in the MCF-7 sensitive cell subline, a time-dependent increase in cells positive for p21WAF1/CIP1 nuclear localization was observed after TGF-beta 1 treatment. These findings suggest that TGF-beta 1 inhibition of MCF-7 cell proliferation is achieved through a type II receptor-dependent down-regulation of Cdk2 kinase activity without modification of Cdk and cyclin expression, but correlated with an increase in p21WAF1/CIP1 nuclear accumulation.