Mediation of c-Myc-induced apoptosis by p53.

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 same characteristics have been described for the tumor suppressor gene p53, the most commonly mutated gene in human cancer. In quiescent mouse fibroblasts expressing wild-type p53 protein, activation of c-Myc was found to induce apoptosis and cell cycle reentry, preceded by stabilization of p53. In contrast, in quiescent p53-null fibroblasts, activation of c-Myc induced cell cycle reentry but not apoptosis. These results suggest that p53 mediates apoptosis as a safeguard mechanism to prevent cell proliferation induced by oncogene activation.

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.

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.

A complex between E2F and the pRb-related protein p130 is specifically targeted by the simian virus 40 large T antigen during cell transformation.

The p130 protein is a recently cloned member of the retinoblastoma protein family. We show here that transformation of NIH3T3-L1 fibroblasts (L1 cells) by the simian virus 40 large T antigen (LTAg) depends on the disruption of DNA binding complexes between transcription factor E2F and p130. LTAg binds to the pocket region of p130 in vivo and disrupts the E2F-p130 complexes. E2F-p130 complexes are present only in quiescent L1 cells and disappear at the G1/S phase boundary concomitantly to induction of DNA synthesis and expression of the E2F-regulated cdc2 gene. p130 is a substrate of cyclin-dependent kinase 2 (Cdk2) in vitro and associates with a Cdk in vivo which is activated upon serum stimulation in late G1. Overexpression of p130 inhibits cdc2 promoter activity and entry of quiescent L1 cells into S phase. The results demonstrate that p130 is negative regulator of cell cycle progression which is specifically targeted by LTAg during cell transformation.

Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work, the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nM of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease S1 analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcription 96 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to cells results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in vivo, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression.

Response of human tumour xenografts to fractionated X-irradiation.

The response of two human tumour xenografts to single dose and fractionated X-rays has been tested using regrowth delay as the assay. The tumours were line transplanted cells from a moderately well-differentiated squamous carcinoma of the tonsillar fossa (XJ) and an undifferentiated carcinoma of the floor of the mouth (XR). Comparison of the dose response curves for single doses in air, clamped, or after misonidazole administration, led to estimates of the hypoxic fraction (approximately 15%) and the sensitizer enhancement ratio (less than or equal to 1.6). When 5 daily fractions were used, the effect of misonidazole (miso) was lost and reoxygenation appeared to be effective in both tumours. Comparison of single doses and 5 fractions in clamped tumours, and in those sensitized by miso, allowed the sparing effect of fractionation to be estimated. When analysed by the linear quadratic model the alpha/beta ratios were found to be in the range of 6.4-9.2 Gy and 6.8-16.0 Gy for the two tumours. These values are in good agreement with murine tumours (assayed in vivo or in vitro), with human tumour cells assayed in vitro, and with analyses of fractionated clinical data for skin cancer.

[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.

[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.

[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.

[Induced astigmatism in cataract surgery. Scleral tunneling incisions of 5.5 mm and 6.5 mm after 1-year follow-up]. / Induzierter Astigmatismus in der Kataraktchirurgie. Skerale Tunnelinzisionen von 5,5 mm und 6,5 mm nach 1jähriger Verlaufskontrolle.

To evaluate the effect of scleral pocket incisions closed with a single horizontal suture on postoperative astigmatism, 97 patients were enrolled in a prospective study. After 1 year of follow-up the data of 80 patients could be analysed. Routine phacoemulsification was performed in all patients consecutively by one surgeon. 40 patients received a posterior chamber lens with a 5 mm by 6 mm oval polymethylmethacrylate (PMMA) optic (group I), and 40 patients received an intraocular lens with a 6 mm diameter round PMMA optic (group II). The incisions were 5.5 mm and 6.5 mm, respectively. Follow-up visits including keratometry were scheduled 1 day, 5 days, 3 months, 6 months and 1 year postoperatively. The induced astigmatism was calculated using vector analysis. One day after operation the mean induced cylinder was 1.22 D in group I and 1.06 D in group II. After 5 days it amounted to 1.09 D (group I) and 1.03 D (group II), and at 3 months it was 1.07 D and 1.00 D, respectively. Six months after operation the induced cylinder was 1.04 D (group I) and 0.96 D (group II), and at 1 year it was 1.02 D and 0.81 D. There was no statistically significant difference between the groups at any time (Wilcoxon test, P > 0.05). We conclude that scleral pocket incisions closed with a single horizontal suture induce about 1 D of corneal astigmatism, with stability over time. There is no clinical advantage in reducing the incision width by using oval optics.

[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.

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.

[Daunomycin in vitrectomy of complicated diabetic retinal detachment]. / Daunomycin bei Vitrektomie komplizierter diabetischer Netzhautablösungen.

Fourteen eyes with combined traction and rhegmatogenous retinal detachments from proliferative diabetic retinopathy were treated with pars plana vitrectomy (PPV) and daunomycin applied to the vitreous cavity using the technique described by Wiedemann et al. for the treatment of proliferative retinopathy. The operations were completed by filling the vitreous with silicone. The aim of our study was to investigate the effect of daunomycin and the reproliferation rate. After a follow-up period of at least 3 months, the post-operative results were compared with a control group of similar eyes treated with PPV and silicone tamponade alone. No evidence was found to the effect that daunomycin affects the rate of reproliferation.

[Sclera fixation of posterior chamber lenses. Indications, technique and results]. / Sklerafixation von Hinterkammerlinsen. Indikationsstellung, Technik und Ergebnisse.

Over 18 months we implanted posterior chamber lenses in 40 eyes using transscleral suturing. The indication for this technique was the absence of capsular support, in most cases due to previous cataract surgery and/or trauma. A uniform procedure was used. After a medium follow-up of 9 months the major complications were as follows: immediately after the operation transient keratopathia was present in a third of the cases; 6 eyes showed iritis, which persisted in 4 eyes. Other late complications were cystoid macular edema in 2 cases and central vein occlusion, retinal detachment and recurrent iritis in single cases. Despite these complications the functional results were favorable in 32 eyes with a best corrected vision of 6/12 or better. Poor visual outcome could be related to the operative procedure in 2 eyes and to pre-existing pathology in the remaining 6 cases. We conclude that the operative technique should be improved to reduce complications.

In vitro study to elucidate the physical laws concerning the fragmentation of both solitary and multiple artificial stones.

These in vitro studies define the basic physical laws regarding work and energy for the successful fragmentation of human gallstones. For this purpose a standardized stone model was used consisting of plaster and glass microspheres with physical properties similar to those of human gallstones. All experiments were performed using the lithotripter model MPL9000 (Dornier). The acoustic energy passing stones of 10-30 mm ranged between 8 and 90 mJ per pulse depending on the stone size and energy setting. These results represent the basis for the three following investigations. In the first experiment the relationship between fragmentation and shock wave energy was investigated in a basket with 2 mm mesh size. Thus no layer of small fragments could shadow the acoustic energy for further fragmentation of larger fragments. A constant amount of stone material was found to be fragmented per shock-wave pulse irrespective of stone volume. A low energy threshold (2 mJ/cm3) was observed, below which fragmentation did not occur. In the second experiment, the sieve was covered with a membrane, thus simulating the in vivo situation. The presence of a layer of small fragments hindered the further disintegration of the larger fragments. The attenuation depended to a large extent on original stone volume and acoustic energy per pulse. The corresponding attenuation factor increased with the original stone volume. Thus the fragmentation of a stone with a diameter of 30 mm was attenuated twice as much as a stone of 20 mm size. The critical layer thickness at which no further disintegration took place was 2.5 mm at 18 kV, 4.2 mm at 22 kV, and 5.0 mm at 26 kV.(ABSTRACT TRUNCATED AT 250 WORDS)

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.