AP4 directly downregulates p16 and p21 to suppress senescence and mediate transformation.

Here we analyzed the function of the c-MYC-inducible basic helix-loop-helix leucine-zipper transcription factor AP4 in AP4-deficient mouse embryo fibroblasts (MEFs). Loss of AP4 resulted in premature senescence and resistance towards immortalization. Senescence was accompanied by induction of the cyclin-dependent kinase inhibitor-encoding genes p16, a known tumor suppressor, and p21, a previously described target for repression by AP4. Notably, AP4 directly repressed p16 expression via conserved E-box motifs in MEFs and human diploid fibroblasts. Senescence caused by AP4-deficiency was prevented by depletion of p16 and/or p21, demonstrating that these factors mediate senescence caused by AP4 loss. As senescence induced by the loss of AP4 was rescued by ectopic AP4, secondary lesions were not involved in causing premature senescence. Activation of c-MYC resulted in repression of p21 and p16 in AP4(+/+), but not in AP4(-/-) MEFs. Furthermore, after combined expression of c-MYC and mutant RAS in MEFs, AP4 was required for colony formation, anchorage-independent growth and tumor formation in mice. In addition, combined ectopic expression of AP4 and mutant RAS in MEFs resulted in colony formation. However, additional loss of the p53 tumor suppressor was necessary for anchorage-independent growth and tumor formation of MEFs by combined AP4 and mutant RAS expression. In conclusion, this study identified AP4 as an oncogenic antagonist of cellular senescence. AP4 achieves this effect by direct repression of p16 and p21, and may thereby critically contribute to c-MYC function and tumor progression.

The miR-34 family in cancer and apoptosis.

Recently, the transcription factor encoded by tumor suppressor gene p53 was shown to regulate the expression of microRNAs. The most significant induction by p53 was observed for the microRNAs miR-34a and miR-34b/c, which turned out to be direct p53 target genes. Ectopic miR-34 expression induces apoptosis, cell-cycle arrest or senescence. In many tumor types the promoters of the miR-34a and the miR-34b/c genes are subject to inactivation by CpG methylation. MiR-34a resides on 1p36 and is commonly deleted in neuroblastomas. Furthermore, the loss of miR-34 expression has been linked to resistance against apoptosis induced by p53 activating agents used in chemotherapy. In this review, the evidence for a role of miR-34a and miR-34b/c in the apoptotic response of normal and tumor cells is surveyed.

The G2/M regulator 14-3-3sigma prevents apoptosis through sequestration of Bax.

In response to DNA damage and genotoxic stress, the p53 tumor suppressor triggers either cell cycle arrest or apoptosis. The G(2) arrest after damage is, in part, mediated by the p53 target, 14-3-3final sigma (final sigma). Colorectal tumor cells lacking final sigma are exquisitely sensitive to DNA damage. Here we analyzed the mechanism of this sensitivity in final sigma(-/-) as compared with final sigma(+/+) human colorectal tumor cells. Exposure to adriamycin resulted in rapid apoptosis only in final sigma(-/-) cells. This was further characterized by caspase-3 activation, p21(CIP1) cleavage, and CDK2 activation. Moreover, Bax was rapidly translocated out of the cytoplasm, and cytochrome c was released in final sigma(-/-) cells. Transient adenovirus-mediated reconstitution of final sigma in the final sigma(-/-) cells led to effective rescue of this phenotype and protected cells against apoptosis. The association of final sigma, Bax, and CDK1 in protein complexes may be the basis for this antiapoptotic mechanism. In conclusion, final sigma not only enforces the p53-dependent G(2) arrest but also delays the apoptotic signal transduction.

Cooperative effects of genes controlling the G(2)/M checkpoint.

It is believed that multiple effectors independently control the checkpoints permitting transitions between cell cycle phases. However, this has not been rigorously demonstrated in mammalian cells. The p53-induced genes p21 and 14-3-3sigma are each required for the G(2) arrest and allow a specific test of this fundamental tenet. We generated human cells deficient in both p21 and 14-3-3sigma and determined whether the double knockout was more sensitive to DNA damage than either single knockout. p21(-/-) 14-3-3sigma(-/-) cells were significantly more sensitive to DNA damage or to the exogenous expression of p53 than cells lacking only p21 or only 14-3-3sigma. Thus, p21 and 14-3-3sigma play distinct but complementary roles in the G(2)/M checkpoint, and help explain why genes at the nodal points of growth arrest pathways, like p53, are the targets of mutation in cancer cells.

High frequency of hypermethylation at the 14-3-3 sigma locus leads to gene silencing in breast cancer.

Expression of 14-3-3 final sigma (final sigma) is induced in response to DNA damage, and causes cells to arrest in G(2). By SAGE (serial analysis of gene expression) analysis, we identified final sigma as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, final sigma mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at final sigma such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the final sigma gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of final sigma is functionally important, because treatment of final sigma-non-expressing breast cancer cell lines with the drug 5-aza-2'-deoxycytidine resulted in demethylation of the gene and synthesis of final sigma mRNA. Breast cancer cells lacking final sigma expression showed increased number of chromosomal breaks and gaps when exposed to gamma-irradiation. Therefore, it is possible that loss of final sigma expression contributes to malignant transformation by impairing the G(2) cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of final sigma expression are the most consistent molecular alterations in breast cancer identified so far.

Identification of CDK4 as a target of c-MYC.

The prototypic oncogene c-MYC encodes a transcription factor that can drive proliferation by promoting cell-cycle reentry. However, the mechanisms through which c-MYC achieves these effects have been unclear. Using serial analysis of gene expression, we have identified the cyclin-dependent kinase 4 (CDK4) gene as a transcriptional target of c-MYC. c-MYC induced a rapid increase in CDK4 mRNA levels through four highly conserved c-MYC binding sites within the CDK4 promoter. Cell-cycle progression is delayed in c-MYC-deficient RAT1 cells, and this delay was associated with a defect in CDK4 induction. Ectopic expression of CDK4 in these cells partially alleviated the growth defect. Thus, CDK4 provides a direct link between the oncogenic effects of c-MYC and cell-cycle regulation.

14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage.

14-3-3Sigma is a member of a family of proteins that regulate cellular activity by binding and sequestering phosphorylated proteins. It has been suggested that 14-3-3sigma promotes pre-mitotic cell-cycle arrest following DNA damage, and that its expression can be controlled by the p53 tumour suppressor gene. Here we describe an improved approach to the generation of human somatic-cell knockouts, which we have used to generate human colorectal cancer cells in which both 14-3-3sigma alleles are inactivated. After DNA damage, these cells initially arrested in the G2 phase of the cell cycle, but, unlike cells containing 14-3-3sigma, the 14-3-3sigma-/- cells were unable to maintain cell-cycle arrest. The 14-3-3sigma-/- cells died ('mitotic catastrophe') as they entered mitosis. This process was associated with a failure of the 14-3-3sigma-deficient cells to sequester the proteins (cyclin B1 and cdc2) that initiate mitosis and prevent them from entering the nucleus. These results may indicate a mechanism for maintaining the G2 checkpoint and preventing mitotic death.

[Late dislocation of the capsular bag after phacoemulsification with endocapsular IOL in pseudoexfoliation syndrome]. / Späte Luxation des Kapselsackes nach Phakoemulsifikation mit endokapsulärer IOL beim Pseudoexfoliationssyndrom.

UNLABELLED: PES has been associated with weakness of zonular attachments to the ciliary body and with late dislocation of the IOL following cataract extraction with in-the-bag IOL implantation. We evaluated the frequency of PES in patients with IOL dislocation following phacoemulsification cataract surgery. PATIENTS AND METHODS: We retrospectively reviewed the records of five patients who had undergone phacoemulsification, followed by implantation of a posterior chamber IOL in the capsular bag during the years 1992 through 1996 and who showed IOL dislocation during follow-up. We looked for clinical signs of PES that had been noted prior to or after IOL dislocation. RESULTS: Detailed review of the records of these patients identified PES as a coexistent condition in all five. The most impressive clinical feature was shrinkage of the capsular bag to conform to the size and contour of the implanted IOL. CONCLUSIONS: In accordance with many histopathological and clinical studies supporting our findings, we suggest that shrinkage of the capsular bag can be reduced by not using foldable IOL, by early anterior YAG capsulotomy or by the use of an IOL with a larger optic.

[Sclera fixation of posterior chamber lenses in vitrectomized eyes. Perfluorocarbon as an operative aid]. / Sklerafixation von Hinterkammerlinsen in vitrektomierten Augen. Perfluorkarbon als operatives Hilfsmittel.

INTRODUCTION: Accurate positioning of transsclerally fixated intraocular lenses in vitrectomized eyes is difficult to achieve because of the lack of stability of the globe. External stabilization with a Flieringa device is widely used. Internal stabilization, however, with liquid perfluorocarbon seems to be more advantageous. METHODS: Endostabilization with perfluorocarbon after pars plana vitrectomy prior to transscleral fixation of a posterior chamber lens was performed in 18 eyes. The intraocular lens floats on the surface of the perfluorocarbon. The level of the liquid can be adjusted to position the lens accurately. Transscleral fixation of the intraocular lens was followed by silicone oil injection in 4 eyes and by BSS injection in 14 eyes. RESULTS AND CONCLUSIONS: Endostabilization of the globe with liquid perfluorocarbon serves as a diaphragm preventing hemorrhage into the vitreous cavity and sliding of the intraocular lens, thus permitting proper positioning. There were no complications either intraoperatively, or postoperatively. Perfluorocarbon is a considerable aid in facilitating the positioning of a transsclerally fixated intraocular lens.

Identification of c-MYC as a target of the APC pathway.

The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.

Physical laws of cholesterol gallstone fragmentation.

Efficient fragmentation is the most important prerequisite for successful treatment of gallstones by extracorporeally induced shock waves. No data are available on the amount of energy necessary for stone disintegration and on the threshold energy below which no further fragmentation occurs. We therefore performed an in vitro investigation on human cholesterol gallstones to elucidate physical laws governing shock-wave lithotripsy. First, the focal pressure of the lithotripter was measured to calculate the energy traversing a stone. Second, 96 gallstones from 16 gall bladders were analysed with respect to physicochemical composition, radiological features and ultrasound before fragmentation was performed. Energy for stone disintegration was constant within each stone family but varied between 4.6 mL-1 and 36.8J mL-1 in different families. This energy correlated linearly with stone volume. None of the radiological and physicochemical factors revealed a clear-cut correlation of the different energies necessary for similar stone disintegration. The threshold energy differed between 0.26 mJ and 1.04 mJ per pulse. In conclusion, stone volume was the best parameter predicting stone fragmentation. However, in cholesterol stones with a similar composition the required energy per volume varies considerably together with the threshold energy. Radiological and ultrasound parameters appear to be of minor importance in explaining these differences.

[Disruption of the blood-aqueous barrier after implantation of sclera-fixed posterior chamber lenses. Early postoperative phase and long-term outcome]. / Beeinträchtigung der Blut-Kammerwasser-Schranke nach Implantation von sklerafixierten Hinterkammerlinsen. Frühe postoperative Phase und Langzeitergebnisse.

The implantation of transsclerally sutured posterior chamber lenses (PCL) leads to greater trauma to the eye than endocapsular PCL implantation. Persistent breakdown of the blood-aqueous barrier might impair the postoperative long-term prognosis. Using laser-tyndallometry, we quantified the disorder of the barrier function during the early postoperative phase and in the long-term postoperative course for both surgical procedures. During the first 3 postoperative days, flare values were three times higher in the group with transsclerally sutured PCL than in the conventional PCL group. Cell counts after transscleral suture fixation (27.7 +/- 18.3/0.075 mm3) decreased slightly during the first 5 postoperative days. In contrast, the conventional PCL group regained the preoperative level (2.5 +/- 5.1) after 3 days. After 3 months, no significant differences in flare values and cell counts were seen between the two groups. After implantation of transsclerally sutured posterior chamber lenses, breakdown of the blood-aqueous barrier was initially more pronounced than in conventional PCL implantation. However, the barrier function was re-established equally in both groups in the long term. No signs of a persistent disorder of the barrier were found.

[Lacrimal duct recanalization with balloon catheter]. / Tränenwegrekanalisation mit Ballonkatheter.

In dilation of nasolacrimal duct stenosis materials now being used originally were developed for percutaneous transluminal coronary angioplasty (PTCA). Suitable modifications of the angioplasty equipment could improve therapeutic results and thus influence the area of indication of this new technique. In addition to the standard PTCA equipment, we used a cannula designed to fit the guide wire, leading to less traumatic intubation of the lacrimal duct and allowing radiographic control of the recanalization procedure. Five patients showing relative stenosis and ten patients showing complete obstruction of the nasolacrimal duct were treated by dacryocystoplasty. Clinical and radiographic follow-up was done over a period of 6 months. In a total of seven patients, dilation proved successful. Out of these, four showed stenosis (80% recanalization rate) and three complete obstruction (30% recanalization rate) before dilation. Recanalization by dilation using balloon catheters seems to be possible even in cases of complete nasolacrimal duct obstruction. However, the success rate is considerably lower than in relative stenosis.

14-3-3sigma is a p53-regulated inhibitor of G2/M progression.

Exposure of colorectal cancer (CRC) cells to ionizing radiation results in a cell-cycle arrest in G1 and G2. The G1 arrest is due to p53-mediated induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/SDI1, but the basis for the G2 arrest is unknown. Through a quantitative analysis of gene expression patterns in CRC cell lines, we have discovered that 14-3-3sigma is strongly induced by gamma irradiation and other DNA-damaging agents. The induction of 14-3-3sigma is mediated by a p53-responsive element located 1.8 kb upstream of its transcription start site. Exogenous introduction of 14-3-3sigma into cycling cells results in a G2 arrest. As the fission yeast 14-3-3 homologs rad24 and rad25 mediate similar checkpoint effects, these results document a molecular mechanism for G2/M control that is conserved throughout eukaryotic evolution and regulated in human cells by p53.

Consensus-interferon and platelet-derived growth factor adversely regulate proliferation and migration of Kaposi's sarcoma cells by control of c-myc expression.

Platelet-derived growth factor-B (PDGF-B) is a potent paracrine-acting mitogen in Kaposi's sarcoma (KS) lesions. Interferon-alpha is widely used for clinical treatment of KS. Here we show that platelet-derived growth factor-B activates proliferation and migration of cultivated AIDS-KS spindle cells whereas interferon-alpha acts as an inhibitor. At the molecular level, these opposite activities of platelet-derived growth factor-B and interferon-alpha converged onto the adverse regulation of the c-myc gene expression. Platelet-derived growth factor-B induced c-myc mRNA and protein synthesis in cultivated AIDS-KS spindle cells whereas interferon-alpha inhibited these processes. Using c-myc-specific phoshothioate antisense oligodeoxynucleotides, we demonstrated that down-regulation of c-myc expression is sufficient to inhibit proliferation and migration of KS spindle cells in vitro. This indicated that c-Myc protein may be an important regulatory molecule of KS spindle cell proliferation and migration. High amounts of the c-Myc protein were detected in the nuclei of KS spindle cells in histological sections of AIDS-KS biopsies. This suggested that the c-myc gene may also regulate proliferation and migration of AIDS-KS spindle cells in vivo. In this case, c-myc may play an important role in the focus of major pathogenic and therapeutic pathways of KS.

Induction of apoptosis by the c-Myc helix-loop-helix/leucine zipper domain in mouse 3T3-L1 fibroblasts.

The cellular proto-oncogene c-myc is involved in cell proliferation and transformation but is also implicated in the induction of programmed cell death (apoptosis). The c-Myc protein is a transcriptional activator with a carboxyl-terminal basic region/helix-loop-helix (HLH)/leucine zipper (LZ) domain. It forms heterodimers with the HLH/LZ protein Max and transactivates gene expression after binding DNA E-box elements. We have studied the phenotype of dominant-negative mutants of c-Myc and Max in microinjection experiments. Max mutants with a deleted or mutated basic region inhibited DNA synthesis in serum-stimulated 3T3-L1 mouse fibroblasts. In contrast, mutants of c-Myc expressing only the basic region/HLH/LZ or HLH/LZ domains rapidly induced apoptosis at low and high serum levels. Co-expression of the HLH/LZ domains of c-Myc and Max failed to do so. We suggest that the c-Myc HLH/LZ domain induces apoptosis by specific interaction with cellular factors different to Max.

Abrogation of p53-induced cell cycle arrest by c-Myc: evidence for an inhibitor of p21WAF1/CIP1/SDI1.

The tumor-suppressor p53 inhibits cell cycle progression by direct transactivation of the p21WAF1/CIP1/SDI1 gene, which encodes a universal inhibitor of cyclin dependent kinases (cdk). The proto-oncogene product c-Myc induces cell cycle progression and, in the absence of survival factors, apoptosis. However, a direct link between the cell cycle machinery and c-Myc has not yet been established. We show that c-Myc has not yet been established. We show that c-Myc abrogates a p53-induced G1-arrest without elevating the expression of cdks or cyclins involved in the G1/S-transition. Instead, the results suggest that c-Myc interferes with the inhibitory action of p21 on cdk/cyclin-complexes by inducing a heat-labile inhibitor of p21. The inactivation of p21 and related cdk-inhibitors may explain several of the oncogenic actions of c-Myc, including the induction of proliferation, immortalisation and the inhibition of differentiation. Modulation of cdk activity by the induction of an inhibitor of cdk-inhibitors represents a novel mechanism of cell cycle regulation in mammalian cells.