Autophagy is of increasing interest as a target for cancer therapy. We find that leucine deprivation causes the caspase-dependent apoptotic death of melanoma cells because it fails to appropriately activate autophagy. Hyperactivation of the RAS-MEK pathway, which is common in melanoma, prevents leucine deprivation from inhibiting mTORC1, the main repressor of autophagy under nutrient-rich conditions. In an in vivo tumor xenograft model, the combination of a leucine-free diet and an autophagy inhibitor synergistically suppresses the growth of human melanoma tumors and triggers widespread apoptosis of the cancer cells. Together, our study represents proof of principle that anticancer effects can be obtained with a combination of autophagy inhibition and strategies to deprive tumors of leucine.

mTOR complex 2 (mTORC2) contains the mammalian target of rapamycin (mTOR) kinase and the Rictor regulatory protein and phosphorylates Akt. Whether this function of mTORC2 is critical for cancer progression is unknown. Here, we show that transformed human prostate epithelial cells lacking PTEN require mTORC2 to form tumors when injected into nude mice. Furthermore, we find that Rictor is a haploinsufficient gene and that deleting one copy protects Pten heterozygous mice from prostate cancer. Finally, we show that the development of prostate cancer caused by Pten deletion specifically in prostate epithelium requires mTORC2, but that for normal prostate epithelial cells, mTORC2 activity is nonessential. The selective requirement for mTORC2 in tumor development suggests that mTORC2 inhibitors may be of substantial clinical utility.

The drug rapamycin has important uses in oncology, cardiology, and transplantation medicine, but its clinically relevant molecular effects are not understood. When bound to FKBP12, rapamycin interacts with and inhibits the kinase activity of a multiprotein complex composed of mTOR, mLST8, and raptor (mTORC1). The distinct complex of mTOR, mLST8, and rictor (mTORC2) does not interact with FKBP12-rapamycin and is not thought to be rapamycin sensitive. mTORC2 phosphorylates and activates Akt/PKB, a key regulator of cell survival. Here we show that rapamycin inhibits the assembly of mTORC2 and that, in many cell types, prolonged rapamycin treatment reduces the levels of mTORC2 below those needed to maintain Akt/PKB signaling. The proapoptotic and antitumor effects of rapamycin are suppressed in cells expressing an Akt/PKB mutant that is rapamycin resistant. Our work describes an unforeseen mechanism of action for rapamycin that suggests it can be used to inhibit Akt/PKB in certain cell types.

The phytochemical indole-3-carbinol (I3C), from cruciferous vegetables such as broccoli, has been shown to elicit a potent anti-proliferative response in human breast cancer cell lines. Treatment of the immortalized human mammary epithelial cell line MCF10A with I3C induced a G1 cell cycle arrest, elevated p53 tumor suppressor protein levels and stimulated expression of downstream transcriptional target, p21. I3C treatment also elevated p53 levels in several breast cancer cell lines that express mutant p53. I3C did not arrest MCF10A cells stably transfected with dominant-negative p53, establishing a functional requirement for p53. Cell fractionation and immunolocalization studies revealed a large fraction of stabilized p53 protein in the nucleus of I3C-treated MCF10A cells. With I3C treatment, phosphatidyl-inositol-3-kinase family member ataxia telangiectasia-mutated (ATM) was phosphorylated, as were its substrates p53, CHK2 and BRCA1. Phosphorylation of p53 at the N-terminus has previously been shown to disrupt the interaction between p53 and its ubiquitin ligase, MDM2, and therefore stabilizing p53. Coimmunoprecipitation analysis revealed that I3C reduced by 4-fold the level of MDM2 protein that associated with p53. The p53-MDM2 interaction and absence of p21 production were restored in cells treated with I3C and the ATM inhibitor wortmannin. Significantly, I3C does not increase the number of 53BP1 foci or H2AX phosphorylation, indicating that ATM is activated independent of DNA double-strand breaks. Taken together, our results demonstrate that I3C activates ATM signaling through a novel pathway to stimulate p53 phosphorylation and disruption of the p53-MDM2 interaction, which releases p53 to induce the p21 CDK inhibitor and a G1 cell cycle arrest.(c) 2005 Wiley-Liss, Inc.

OBJECTIVES: To perform a retrospective study to assess the efficacy and safety of electrocauterization of bleeding points after stone extraction using the data from 249 percutaneous nephrolithotomies. METHODS: A total of 341 percutaneous nephrolithotomies in 324 patients were performed at the Chiayi Christian Hospital from July 2000 to July 2003. Electrocauterization of bleeding points with an elongated electrode probe was performed in 249 patients. The age, height, weight, preoperative hemoglobin level, stone burden, operating time, stone free rate, length of postoperative hospital stay, postoperative urinary tract infection rate, and blood transfusion rate were recorded by retrospective chart review. RESULTS: No statistically significant differences in age, height, weight, stone burden, operating time, stone free rate, or length of postoperative hospital stay were found between patients with or without electrocauterization. No increase occurred in the postoperative urinary tract infection rate in patients who received electrocauterization, and these patients had a statistically significant decrease in the transfusion rate. No nephrostomy tube was inserted at the completion of surgery in 84 (33.7%) of the 249 operations in which electrocauterization was performed. CONCLUSIONS: Electrocauterization of the bleeding points at the end of percutaneous renal surgery decreases the blood transfusion rate without causing an increase in morbidity. This procedure is safe and effective and may make more patients suitable for tubeless modification.

BACKGROUND AND PURPOSE: Percutaneous nephrolithotomy (PCNL) is now a popular method for removal of renal and ureteral stones. Placement of a nephrostomy tube after the completion of PCNL has been considered a standard procedure by most urologists, but some authors have recently challenged this practice. Bleeding is one of the most prevalent problems after nephrostomy tube-free percutaneous renal surgery. To diminish the possibility of postoperative bleeding, we cauterized the PCNL tract to make it bloodless. The efficacy and safety of this procedure were reviewed in this study. PATIENTS AND METHODS: From March 2001 to March 2003, 51 patients underwent PCNL with a one-stage procedure and a single access tract. The stone size ranged from 1.0 to 7.0 cm (mean 2.7 +/- 1.4 cm). A holmium:YAG laser and pneumatic lithotripter were used. After stone extraction, a 6F double-J catheter was inserted antegrade. The access tract was checked, and the bleeding points were cauterized. No nephrostomy tube was inserted, but a Penrose drain was left overnight. Perforation of the collecting system was not a contraindication to tubeless PCNL. RESULTS: The stone-free rate was 80.4%, including five patients with complete staghorn stones. Twenty-one patients required postoperative analgesics. Only one patient had urine leakage for longer than 24 hours. Transient low fever was noted in five patients, but no patient experienced severe urinary tract infection. Delayed hemorrhage (1 week after the operation) secondary to irritation by the double-J ureteral stent was noted in one patient. The average postoperative hospital stay was 2.2 days (range 1-3 days). No patient required a blood transfusion. No urinoma was noted on the postoperative ultrasound follow-up. CONCLUSION: Nephrostomy tube-free percutaneous renal surgery is a safe and effective procedure for selected patients with minimal hemorrhage after PCNL. Cauterization of tract bleeding points may make this modification a more secure procedure and make it suitable for more patients.

Effects of c-Myc overexpression on the DNA damage-induced G2/M checkpoint were studied in finite lifespan, normal human mammary epithelial cells (HMECs). Previously, we showed that c-Myc attenuates G1/S arrest and leads to an inappropriate entry of cells with damaged DNA into the S phase, following treatment with ionising radiation (IR). Here we show that, in striking contrast to control cells, c-Myc-overexpressing HMECs demonstrate a significant attenuation of the G2/M arrest, following IR, and enter into inappropriate mitoses. At the molecular level, ectopic overexpression of c-Myc leads to an unusually high level of expression of cyclin B1, and the elevated levels of cyclin B1 were maintained, after gamma-irradiation. Introduction of DNA damage in c-Myc-overexpressing, normal mammary epithelial cells eventually induces apoptosis, indicating a dramatic sensitisation by c-Myc of DNA damage-induced apoptosis. These two remarkable phenotypes, checkpoint attenuation and sensitisation to apoptosis, resulting from a deregulation of the protooncogene c-myc, may produce a unique pattern of alternating cycles, consisting first of amplification of DNA damage, followed by apoptosis-assisted selective pressure. The result of this alternating pattern of damage apoptosis could facilitate the selection of certain genomic alterations required for cellular survival and cellular transformation.

Study of the mechanism(s) of genomic instability induced by the c-myc proto-oncogene has the potential to shed new light on its well-known oncogenic activity. However, an underlying mechanism(s) for this phenotype is largely unknown. In the present study, we investigated the effects of c-Myc overexpression on the DNA damage-induced G(1)/S checkpoint, in order to obtain mechanistic insights into how deregulated c-Myc destabilizes the cellular genome. The DNA damage-induced checkpoints are among the primary safeguard mechanisms for genomic stability, and alterations of cell cycle checkpoints are known to be crucial for certain types of genomic instability, such as gene amplification. The effects of c-Myc overexpression were studied in human mammary epithelial cells (HMEC) as one approach to understanding the c-Myc-induced genomic instability in the context of mammary tumorigenesis. Initially, flow-cytometric analyses were used with two c-Myc-overexpressing, nontransformed immortal lines (184A1N4 and MCF10A) to determine whether c-Myc overexpression leads to alteration of cell cycle arrest following ionizing radiation (IR). Inappropriate entry into S phase was then confirmed with a bromodeoxyuridine incorporation assay measuring de novo DNA synthesis following IR. Direct involvement of c-Myc overexpression in alteration of the G(1)/S checkpoint was then confirmed by utilizing the MycER construct, a regulatable c-Myc. A transient excess of c-Myc activity, provided by the activated MycER, was similarly able to induce the inappropriate de novo DNA synthesis following IR. Significantly, the transient expression of full-length c-Myc in normal mortal HMECs also facilitated entry into S phase and the inappropriate de novo DNA synthesis following IR. Furthermore, irradiated, c-Myc-infected, normal HMECs developed a sub-G(1) population and a >4N population of cells. The c-Myc-induced alteration of the G(1)/S checkpoint was also compared to the effects of expression of MycS (N-terminally truncated c-Myc) and p53DD (a dominant negative p53) in the HMECs. We observed inappropriate hyperphosphorylation of retinoblastoma protein and then the reappearance of cyclin A, following IR, selectively in full-length c-Myc- and p53DD-overexpressing MCF10A cells. Based on these results, we propose that c-Myc attenuates a safeguard mechanism for genomic stability; this property may contribute to c-Myc-induced genomic instability and to the potent oncogenic activity of c-Myc.

Over the past 35 years, aesthetic rhinoplasty has evolved from a generic, reductive operation to a highly individualized, problem-specific operation that often combines augmentation with reduction. The author's experience has been marked by the following conceptual and technical milestones that have contributed to an ongoing exploration and advancement of nasal surgery:(1) vestibular stenosis: diagnosis of a surgical consequence;(2) etiology and treatment of supratip deformity: the dynamic relationship of soft-tissue contour to skeleton;(3) etiology and treatment of the tip with inadequate projection: tip graft design;(4) practical aesthetics of balance: the augmentation-reduction approach to rhinoplasty;(5) support of the middle vault: functional and aesthetic effects;(6) malposition of the lateral crura: recognition and management; and (7) the significance of the middle crura: clinical and aesthetic considerations.