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Purpose: Cervical tumor response on posttherapy 2[(18)F]fluoro-2-deoxy-d-glucose-positron emission tomography (FDG-PET) is predictive of survival outcome. The purpose of this study was to use gene expression profiling to identify pathways associated with tumor metabolic response., Experimental Design: This was a prospective tissue collection study for gene expression profiling of 62 pretreatment biopsies from patients with advanced cervical cancer. Patients were treated with definitive radiation. Fifty-three patients received concurrent chemotherapy. All patients underwent a pretreatment and a 3-month posttherapy FDG-PET/computed tomography (CT). Tumor RNA was harvested from fresh frozen tissue and hybridized to Affymetrix U133Plus2 GeneChips. Gene set enrichment analysis (GSEA) was used to identify signaling pathways associated with tumor metabolic response. Immunohistochemistry and in vitro FDG uptake assays were used to confirm our results., Results: There were 40 biopsies from patients with a complete metabolic response (PET-negative group) and 22 biopsies from patients with incomplete metabolic response (PET-positive group). The 3-year cause-specific survival estimates were 98% for the PET-negative group and 39% for the PET-positive group (P < 0.0001). GSEA identified alterations in expression of genes associated with the PI3K/Akt signaling pathway in patients with a positive follow-up PET. Immunohistochemistry using a tissue microarray of 174 pretreatment biopsies confirmed p-Akt as a biomarker for poor prognosis in cervical cancer. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 inhibited FDG uptake in vitro in cervical cancer cell lines., Conclusions: Activation of the PI3K/Akt pathway is associated with incomplete metabolic response in cervical cancer. Targeted inhibition of PI3K/Akt may improve response to chemoradiation.

Objective: The objective of this study was to investigate the chronic toxicity, response to therapy, and survival outcomes of patients with cervical cancer treated with definitive pelvic irradiation delivered by helical tomotherapy (HT), with or without concurrent chemotherapy., Methods and Materials: There were 15 patients with a new diagnosis of cervical cancer evaluated in this study from April 2006 to February 2007. The clinical stages of their disease were Stage Ib1 in 3 patients, Ib2 in 3, IIa in 2, IIb in 4, IIIb in 2, and IVa in 1 patient. Fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) simulation was performed in all patients. All patients received pelvic irradiation delivered by HT and high-dose-rate (HDR) brachytherapy. Four patients also received para-aortic irradiation delivered by HT. Thirteen patients received concurrent chemotherapy. Patients were monitored for chronic toxicity using the Common Terminology Criteria for Adverse Events version 3.0 criteria., Results: The median age of the cohort was 51 years (range, 29-87 years), and the median follow-up for all patients alive at time of last follow-up was 35 months. The median overall radiation treatment time was 54 days. One patient developed a chronic Grade 3 GI complication. No other Grade 3 or 4 complications were observed. At last follow-up, 3 patients had developed a recurrence, with 1 patient dying of disease progression. The 3-year progression-free and cause-specific survival estimates for all patients were 80% and 93%, respectively., Conclusion: Intensity-modulated radiation therapy delivered with HT and HDR brachytherapy with or without chemotherapy for definitive treatment of cervical cancer is feasible, with acceptable levels of chronic toxicity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: To investigate, in a prospective trial, the acute and chronic toxicity of patients with cervical cancer treated with surgery and postoperative intensity-modulated radiotherapy (RT) delivered using helical tomotherapy, with or without the administration of concurrent chemotherapy., Patients and Methods: A total of 24 evaluable patients entered the study between March 2006 and August 2009. The indications for postoperative RT were tumor size, lymphovascular space invasion, and the depth of cervical stromal invasion in 15 patients; 9 patients underwent postoperative RT because of surgically positive lymph nodes. All patients underwent pelvic RT delivered with helical tomotherapy and intracavitary high-dose-rate brachytherapy. Treatment consisted of concurrent weekly platinum in 17, sequential carboplatin/Taxol in 1, and RT alone in 6. The patients were monitored for acute and chronic toxicity using the Common Toxicity Criteria, version 3.0., Results: The median follow-up was 24 months (range, 4-49). At the last follow-up visit, 23 patients were alive and disease free. Of the 24 patients, 12 (50%) experienced acute Grade 3 gastrointestinal toxicity (anorexia in 5, diarrhea in 4, and nausea in 3). One patient developed acute Grade 4 genitourinary toxicity (vesicovaginal fistula). For patients treated with concurrent chemotherapy, the incidence of acute Grade 3 and 4 hematologic toxicity was 71% and 24%, respectively. For patients treated without concurrent chemotherapy, the incidence of acute Grade 3 and 4 hematologic toxicity was 29% and 14%, respectively. Two long-term toxicities occurred (vesicovaginal fistula at 25 months and small bowel obstruction at 30 months). The overall and progression-free survival rate at 3 years for all patients was 100% and 89%, respectively., Conclusion: The results of our study have shown that postoperative external RT for cervical cancer delivered with helical tomotherapy and high-dose-rate brachytherapy and with or without chemotherapy is feasible, with acceptable acute and chronic toxicity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Squamous cell carcinoma antigen (SCC Ag) is a serum biomarker for squamous cell carcinoma (SCC) of the cervix. We investigated the prognostic significance of SCC Ag levels before and at the completion of chemoradiotherapy and compared these levels with the results of pre- and posttreatment positron emission tomography/computed tomography (PET/CT) using [(18)F]fluorodeoxyglucose (FDG)., Methods and Materials: The records of 63 women who underwent definitive chemoradiotherapy for SCC of the cervix were reviewed. SCC Ag levels were obtained before and at the completion of radiotherapy. Patients were divided into two groups on the basis of their pretreatment SCC Ag level (>30 ng/mL vs. ≤30 ng/mL). Pre- and posttreatment FDG-PET/CT characteristics and progression-free survival (PFS) were analyzed according to SCC Ag groups., Results: Median follow-up was 12 months. Women with SCC Ag >30 ng/mL at diagnosis had more advanced lymph node disease on pretreatment FDG-PET/CT than those with SCC Ag ≤30 ng/mL (p = .002). Women whose SCC Ag normalized at the completion of chemoradiotherapy were more likely to have a complete metabolic response on their 3-month posttreatment FDG-PET/CT than those whose SCC Ag did not normalize (p = .006). The 2-year PFS was 73% for patients with a SCC Ag level ≤30 ng/mL at diagnosis compared with 0% for those with a SCC Ag level >30 ng/mL at diagnosis (p < .0001). The 2-year PFS was 62% for patients whose SCC Ag normalized at the completion of chemoradiotherapy compared with 0% for those whose SCC Ag did not normalize (p = .0004)., Conclusion: Elevated SCC Ag at diagnosis and failure of the SCC Ag to normalize at the completion of treatment are associated with incomplete metabolic response and decreased PFS., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Invasive cervical cancer is a leading cause of cancer death in women worldwide, resulting in about 300,000 deaths each year. The clinical outcomes of cervical cancer vary significantly and are difficult to predict. Thus, a method to reliably predict disease outcome would be important for individualized therapy by identifying patients with high risk of treatment failures before therapy. In this study, we have identified a microRNA (miRNA)-based signature for the prediction of cervical cancer survival. miRNAs are a newly identified family of small noncoding RNAs that are extensively involved in human cancers. Using an established PCR-based miRNA assay to analyze 102 cervical cancer samples, we identified miR-200a and miR-9 as two miRNAs that could predict patient survival. A logistic regression model was developed based on these two miRNAs and the prognostic value of the model was subsequently validated with independent cervical cancers. Furthermore, functional studies were done to characterize the effect of miRNAs in cervical cancer cells. Our results suggest that both miR-200a and miR-9 could play important regulatory roles in cervical cancer control. In particular, miR-200a is likely to affect the metastatic potential of cervical cancer cells by coordinate suppression of multiple genes controlling cell motility.

Objective: Ovarian cancer is one of the most deadly human cancers, resulting in over 15,000 deaths in the US each year. A reliable method that could predict disease outcome would improve care of patients with this disease. The main aim of this study is to identify novel prognostic biomarkers for advanced ovarian cancer., Methods: We hypothesized that microRNAs (miRNAs) may predict outcome and have examined the prognostic value of these small RNA molecules on disease outcome prediction. miRNAs are a newly identified family of non-coding RNA genes, and recent studies have shown that miRNAs are extensively involved in the tumor development process. We have profiled the expression of miRNAs in advanced ovarian cancer using a novel PCR-based platform and correlated miRNA expression profiles with disease outcome., Results: By performing miRNA expression profiling analysis of 55 advanced ovarian tumors, we have shown that three miR-200 miRNAs (miR-200a, miR-200b and miR-429) in the miR-200b-429 cluster are significantly associated with cancer recurrence and overall survival. Further target analysis indicates that these miR-200 miRNAs target multiple genes that are involved in cancer development. In addition, we have also shown that overexpression of this miR-200 cluster inhibits ovarian cancer cell migration., Conclusions: miR-200b-429 may be used as a prognostic marker for ovarian cancer outcome, and low-level expression of miR-200 miRNAs in this cluster predicts poor survival. In addition, our study suggests that miR-200 miRNAs could play an important regulatory role in ovarian cancer.

For locally advanced cervical cancer, the current literature supports the use of (18)F-FDG PET for assessing treatment response 3 mo after the completion of concurrent chemoradiation. (18)F-FDG PET can provide reliable long-term prognostic information for these patients and, in the future, may be used to guide additional therapy. Investigational areas include the use of (18)F-FDG PET for monitoring response during radiotherapy and chemotherapy in the metastatic and neoadjuvant settings. For ovarian masses, the performance of (18)F-FDG PET in the detection of borderline tumors is limited, and the presence of physiologic (18)F-FDG uptake in normal ovaries of premenopausal women poses another limitation. Preliminary data suggest that the performance of (18)F-FDG PET and (18)F-FDG PET/CT is superior to that of CT alone in initial staging, but the sensitivity of both in the detection of carcinomatosis is limited. Preliminary data also suggest that (18)F-FDG PET may be promising for early prediction of response to chemotherapy and for prediction of response after the completion of chemotherapy. (18)F-FDG PET and (18)F-FDG PET/CT are most helpful in the evaluation of patients with suspected recurrent ovarian carcinoma, especially when CA-125 levels are rising and CT findings are normal or equivocal. PET and CT are complementary, and PET/CT should be used when available. Preliminary data suggest that the addition of (18)F-FDG PET/CT to the evaluation of these patients changes management in approximately a third and reduces overall treatment costs by accurately identifying patients who will or will not benefit from surgery.

We had previously demonstrated that low dose irinotecan (CPT-11) leads to increased accumulation of cells in S-phase and shows a therapeutic synergy with S-phase specific chemotherapy such as gemcitabine and 5-fluorouracil. In this phase II study, our objectives were to evaluate the tolerability and activity of low dose CPT-11 followed 24 h later by gemcitabine as second line therapy in patients with metastatic non-small cell lung cancer (NSCLC). CPT-11 (60 mg/m) was administered 24 h before gemcitabine (1000 mg/m) on days 1, 2, 8, and 9 every 3 weeks. Twenty-nine patients were evaluable for response. The median follow-up was 7.4 months. Partial response (PR) was seen in two (6.9, 95% confidence interval (CI): 0.009-0.228). PR and stable disease were seen in 22 patients (75.9, 95% CI: 0.564-0.897). The median survival time was 13.8 months (95% CI: 8.1-19.3). The median time to progression was 4.6 months (95% CI: 2.6-6.2). Thirty-eight patients were evaluable for toxicity. Neutropenia (grade 3 or 4) was observed in 27 patients (71%). Eight patients did not receive cycle 2 of therapy owing to prolonged neutropenia. No treatment-related deaths occurred. Scheduled administration of low dose CPT-11, 24 h before gemcitabine in the second line therapy of NSCLC yielded comparable disease control rates (PR and stable disease) when compared with other studies using the two chemotherapy drugs in the traditional sequence. However, this approach was associated with higher grade 3/4 neutropenia and is not recommended for further study in metastatic NSCLC.

Ispinesib (SB-715992) inhibits the mitotic kinesin spindle protein (KSP), a novel target for anticancer therapy. A phase II study was conducted to examine the efficacy of ispinesib in recurrent or metastatic head and neck squamous cell carcinoma (RMHNSC). Patients with up to one prior line of chemotherapy for RMHNSC were treated with ispinesib 18 mg/m2 IV over 1 hour every 21 days. Twenty-one patients were enrolled onto this study with a target stage I sample size of 19. Of 20 evaluable patients, no objective responses were seen and stable disease > 2 cycles was observed in five patients (25%). The median time to progression was 1.4 (95% CI 1.3-2.3) months, median survival was 3.5 (95% CI 2.8-7.8) months, and 1 year overall survival was 20% (95% CI 8.3-48.1%). The most frequent attributable grades III-V adverse events were neutropenia (60% of patients) and leukopenia (55%). The pharmacokinetic profile was consistent with results from phase I studies. Archival tissues (n = 14) demonstrated low to moderate KSP expression by immunohistochemistry. In addition, no pharmacodynamic changes were observed in peripheral blood mononuclear cells. We detected no antitumor activity of ispinesib in RMHNSC on this dosing schedule.

Purpose: To evaluate the response to therapy for anal carcinoma using post-therapy imaging with positron emission tomography (PET)/computed tomography and F-18 fluorodeoxyglucose (FDG) and to compare the metabolic response with patient outcome., Patients and Methods: This was a prospective cohort study of 53 consecutive patients with anal cancer. All patients underwent pre- and post-treatment whole-body FDG-PET/computed tomography. Patients had been treated with external beam radiotherapy and concurrent chemotherapy. Whole-body FDG-PET was performed 0.9-5.4 months (mean, 2.1) after therapy completion., Results: The post-therapy PET scan did not show any abnormal FDG uptake (complete metabolic response) in 44 patients. Persistent abnormal FDG uptake (partial metabolic response) was found in the anal tumor in 9 patients. The 2-year cause-specific survival rate was 94% for patients with a complete vs. 39% for patients with a partial metabolic response in the anal tumor (p = 0.0008). The 2-year progression-free survival rate was 95% for patients with a complete vs. 22% for patients with a partial metabolic response in the anal tumor (p < 0.0001). A Cox proportional hazards model of survival outcome indicated that a complete metabolic response was the most significant predictor of progression-free survival in our patient population (p = 0.0003)., Conclusions: A partial metabolic response in the anal tumor as determined by post-therapy FDG-PET is predictive of significantly decreased progression-free and cause-specific survival after chemoradiotherapy for anal cancer.

Context: Retrospective studies have demonstrated that the use of positron emission tomography (PET) with F-18 fluorodeoxyglucose (FDG) in the posttherapy evaluation of patients with cervical carcinoma is predictive of survival outcome., Objective: To validate the association between the metabolic response on the 3-month posttherapy FDG-PET and long-term survival outcome., Design, Setting, and Patients: A prospective cohort study designed to validate our previous finding that the results of a 3-month posttherapy FDG-PET are predictive of long-term clinical outcome. A total of 92 women were treated with external irradiation, brachytherapy, and concurrent chemotherapy from January 2003 through September 2006. Posttherapy whole-body FDG-PET was performed 2 to 4 months (mean, 3 months) after completion of therapy., Main Outcome Measures: The primary outcome end points were metabolic response, progression-free survival, and cause-specific survival., Results: Posttherapy FDG-PET showed a complete metabolic response in 65 patients (70%), a partial metabolic response in 15 (16%), and progressive disease in 12 (13%). Their 3-year progression-free survival rates were 78%, 33%, and 0%, respectively (P < .001). Multivariate analysis demonstrated that the hazard ratio (HR) for risk of recurrence based on the posttherapy metabolic response showing progressive disease was 32.57 (95% confidence interval [CI], 10.22-103.82). A partial metabolic response had an HR of 6.30 (95% CI, 2.73-14.56). These were more predictive of survival outcome than the pretreatment lymph node status (HR, 3.54; 95% CI, 1.54-8.09)., Conclusion: In this single-site study population of women with cervical cancer, 3-month posttherapy FDG uptake, as detected by whole-body PET, was predictive of survival.

Purpose: To determine if hypoxia-related molecular markers are associated with (60)Cu labeled diacetyl-bis (N4 -methylthiosemicarbazone); ((60)Cu-ATSM) imaging of tumor hypoxia in cervical cancer., Procedures: Fifteen patients were enrolled in a prospective study and underwent evaluation of tumor hypoxia with positron emission tomography (PET) using (60)Cu-ATSM. (60)Cu-ATSM-PET imaging was compared with the expression of tissue molecular markers, which included vascular endothelial growth factor (VEGF), cyclo-oxygenase-2 (COX-2), epidermal growth factor receptor (EGFR), carbonic anyhdrase IX (CA-9), and apoptotic index., Results: Six patients had hypoxic tumors determined by (60)Cu-ATSM, and nine had non-hypoxic tumors. The 4-year overall survival estimates were 75% for patients with non-hypoxic tumors and 33% for those with hypoxic tumors (p = 0.04). Overexpression of VEGF (p = 0.13), EGFR (p = 0.05), CA-9 (p = 0.02), COX-2 (p = 0.08), and the presence of apoptosis (p = 0.005) occurred in patients with hypoxic tumors. Cox proportional hazards modeling demonstrated hypoxia as determined by (60)Cu-ATSM to be a significant independent predictor of tumor recurrence (p = 0.0287)., Conclusions: (60)Cu-ATSM hypoxia was correlated with overexpression of VEGF, EGFR, COX-2, CA-9, an increase in apoptosis, and a poor outcome.

Purpose: Docetaxel, cisplatin, and capecitabine are three active chemotherapeutic agents with different mechanisms of action. This phase I study investigated the feasibility and pharmacokinetics of this combination given on a weekly schedule., Experimental Design: Docetaxel and cisplatin were given i.v. over 30 minutes on days 1 and 8 and capecitabine was given orally bid on days 1 to 14 (every 21 days). Escalation occurred in cohorts of three patients until the maximum tolerated dose was defined. Pharmacokinetics studies of docetaxel and total and ultrafiltrate platinum after cisplatin administration were done on cycle 1 (with capecitabine) and cycle 2 (without capecitabine)., Results: Twenty-five patients were enrolled. Two of six patients at dose level 5 had a dose-limiting infection and diarrhea. One of six evaluable patients at dose level 4 (27 mg/m2 docetaxel, 27 mg/m2 cisplatin, 825 mg/m2 capecitabine) had a dose-limiting hypomagnesemia. Pharmacokinetics of docetaxel were similar on cycles 1 and 2. Area under the plasma concentrations versus time curves of total platinum was significantly greater in cycle 2 compared with cycle 1 (P = 0.001). There was no difference in the disposition of docetaxel on cycles 1 and 2., Conclusions: The recommended docetaxel, cisplatin, and capecitabine dose for phase II studies is 27/27/825 mg/m2. The alteration in total and ultrafiltrate platinum disposition on cycle 2 compared with cycle 1 may be inherent to sequential cisplatin administration; however, prior treatment with capecitabine cannot be ruled out as a factor.

Background: Cutaneous mesothelioma is rare but may occur following local surgical procedures for visceral mesothelioma or as a metastasis., Methods: A patient with pleural mesothelioma, who developed papules within chest wall scars, 14 and 15 months after pleural biopsy and thoracentesis, respectively, is reported., Results: Histopathology showed an epithelioid tumor forming tubulopapillary and glandular structures. The diagnosis of mesothelioma was confirmed with immunohistochemistry. Tumor cells stained characteristically for low-molecular-weight cytokeratins 5/6, calretinin, and vimentin and were negative for mucicarmine, carcinoembryonic antigen, thyroid transcription factor 1, prostate-specific antigen, gross cystic disease fluid protein, S-100, factor VIII, and CD31., Conclusions: Histologically, mesothelioma may resemble a primary adnexal neoplasm, metastatic adenocarcinoma, or angiosarcoma. Immunohistochemistry can clarify the diagnosis. Clinicians should be aware of the varied presentations of mesothelioma, as cutaneous presentations are becoming increasingly common.

Little is known about how American nurses understand and respond to requests made by decisionally capable patients for assistance in dying. This article is based on a broader qualitative study first reported elsewhere (Schwarz, 2003). The study used phenomenological interpretation and analysis of stories told by 10 nurses who worked in home hospice, critical care, and HIV/AIDS care settings. Persistent requests for assistance in dying were relatively uncommon, but when heard, participants provided the following responses: refusing assistance, administering palliative drugs that might secondarily hasten dying, tacitly permitting and not interfering with patient or family plans to hasten death, and actively providing direct assistance in dying. Nurses' responses were context-driven; they did not seek guidance from professional codes of ethics or colleagues. Secrecy and collusion were routinely practised. Few participants unequivocally agreed or refused to help patients die; most struggled to find morally and legally acceptable ways to help patients die well. Regardless of how they responded, nurses who believed they had hastened death described feelings of guilt and moral distress. Healthcare professionals who provide care for symptomatic dying patients need opportunities to meet with supportive colleagues, to share the experience of troubling cases and of moral conflict, and to be supported and heard in a 'safe' environment.

Treatment strategies that have the potential to improve functional organ preservation in patients who have head and neck cancer are emerging. Clinical research in this field, however, has been limited by the lack of standardized, objective criteria of organ function post treatment and by lack of prospective assessment of organ function in treatment trials [56]. Advances in surgical techniques, radiation techniques, radiation protectants, and combined-modality therapies are promising, but well-planned and executed clinical trials are necessary to determine how best to apply these techniques to patient care.

Chk2 is a serine/threonine protein kinase found mutated in certain hereditary and sporadic cancers. Ionizing radiation (IR) activates the kinase activity of Chk2 in a phosphorylation-dependent manner. ATM phosphorylates Chk2 on threonine 68, which promotes oligomerization and phosphorylation on threonines 383 and 387 within the activation loop of the catalytic domain. In this study, threonines 68, 383, and 387 were confirmed as sites of Chk2 phosphorylation both in vitro and in vivo. In addition, serine 516 was identified as a novel IR-inducible phosphorylation site in vivo and as a site of autophosphorylation in vitro. Interestingly, Chk2 was capable of autoactivation in the absence of IR when overproduced in bacteria, in 293 cells, and in murine embryonic fibroblasts lacking Chk2. A kinase-inactive mutant of Chk2 was phosphorylated on T68 and T383/T387 but not on S516 in cells containing Chk2 and on T68 but not T383/T387 or S516 in cells lacking Chk2. This establishes a dependency on Chk2 kinase activity for phosphorylation of T383/T387 and S516 but not for T68 in vivo. We demonstrate that T68 phosphorylation is regulated by kinases in addition to ATM and Chk2. Taken together, our data indicate that autophosphorylation of Chk2 can occur both in cis and in trans and suggest that oligomerization may regulate Chk2 activation by promoting these cis- and trans-phosphorylation events. The importance of oligomerization is underscored by the observation that substitution of isoleucine for threonine at position 157, a mutation found in a subset of patients with Li-Fraumeni syndrome, impairs both Chk2 oligomerization and autophosphorylation.

Purpose: To explore how nurses experience and respond to patients' requests for assistance in dying (AID)., Design and Methods: A phenomenological study of 10 self-selected nurses., Findings: Four major themes: Being Open to Hear and Hearing; Interpreting and Responding to the Meaning; Responding to Persistent Requests for AID, and Reflections. When faced with persistent requests for AID, participants provided a continuum of interventions: refusal, providing palliative care that might secondarily hasten dying, respecting and not interfering with patients' or families' plans to hasten dying, and providing varying types and degrees of direct AID. Their responses were context-driven rather than rule-mandated, and they drew a distinction between secondarily hastening and directly causing death., Conclusions: Few nurses in this study unequivocally agreed or refused to directly help a patient die. Most struggled alone and in silence to find a morally and legally acceptable way to help patients who persisted in requesting AID. Regardless of how they responded, many described feelings of conflict, guilt, and moral distress.

Little is currently known about the context, nature, or frequency of nurses' responses to patient requests for help in dying. Only two empirical studies have surveyed American nurses about their actual responses to such requests. In one of those studies, 441 New England oncology nurses described how often patients ask them for help in ending their lives and also indicated how often they participated in assisted suicide and patient-requested euthanasia. One hundred and ten of those 441 nurses wrote comments on their returned questionnaires. This article describes the content analysis of those comments. Those oncology nurses who wrote have much to say about caring for patients at the end of life., (Copyright 2001 by W.B. Saunders Company)

Eukaryotic cells activate an evolutionarily conserved set of proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed. In response to ionizing radiation (IR), the cell cycle checkpoint kinase, Chk2 (hCds1), is phosphorylated and activated in an ataxia telangiectasia mutated (ATM)-dependent manner. Here we show that the ATM protein kinase directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and that T68 is phosphorylated in vivo in response to IR in an ATM-dependent manner. Furthermore, phosphorylation of T68 was required for full activation of Chk2 after IR. Together, these data are consistent with the model that ATM directly phosphorylates Chk2 in vivo and that this event contributes to the activation of Chk2 in irradiated cells.

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.

Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe (Sp), SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. Here we report the identification of a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural, and functional similarity to SpCds1. HuCds1 was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after hydroxyurea treatment. Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins. These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals.

Purpose: Nurses' views are often solicited about physician-assisted dying, a concept that incorporates both assisted suicide and active euthanasia. Yet nurses are rarely asked about their own clinical experience of assisted dying. The literature indicates that many nurses experience difficulty distinguishing professionally sanctioned end-of-life interventions from those that are not. In this article the investigator explores the social, legal, and political roots of assistance in dying, and critically examines the profession's position on nurse participation in assisted dying and the research regarding nurse-assisted dying., Scope: The bioethics and nursing literature was reviewed from 1990 to 1999. The databases used were the Cumulative Index to Nursing and Allied Health Literature and Medline., Conclusions: The complex nature of caring for highly symptomatic dying patients, and the difficulty some nurses experience in distinguishing a moral difference between hastening and assisting death, strongly indicate a need for additional nursing research that does not use a forced answer.

Transport of L-lysine by a cultured placental trophoblast cell line was investigated by characterization of L-[3H]lysine uptake. In the mononuclear form of the BeWo clone b30 choriocarcinoma cell, at least two sodium-independent systems are present. Concentration dependence data were fitted by a two system model with Km values (+/- s.e.) of 2 +/- 0.7 and 94 +/- 31 microM and Vmax values (+/- s.e.) of 0.7 +/- 0.3 and 25 +/- 6.0 nM/mg DNA/min. A portion of sodium-independent uptake was inhibited by the sulphydryl modifying reagent N-ethylmaleimide (NEM). Following NEM treatment, the data were fitted by a single system with Km = 10 +/- 2 microM AND Vmax = 5.1 +/- 0.8 nM/mg DNA/min. In the absence of sodium, NEM-resistant uptake was sensitively inhibited by leucine whereas NEM-sensitive uptake was not inhibited by leucine. It is concluded that like placental basal membrane, the mononuclear BeWo cell possesses two sodium-independent L-lysine transport systems. The high-capacity, NEM-sensitive, leucine-insensitive system resembles the widespread system y+. The high-affinity, NEM-resistant, leucine-sensitive system resembles system b(0,+).

We have tested the suitability of the green fluorescent protein (GFP) of Aequorea victoria as a marker for studies of gene expression and protein targeting in the trypanosomatid parasite Leishmania. Leishmania promastigotes expressing GFP from episomal pXG vectors showed a bright green fluorescence distributed throughout the cell, readily distinguishable from control parasites. Transfection of a modified GFP gene containing GC-rich synonymous codons and the S65T mutation (GFP+) yielded a much higher fluorescence. FACS analysis revealed a clear quantitative separation between GFP-transfected and control parasites, with pXG-GFP+ transfectants showing fluorescence signals more than 100-fold background. Episomal DNAs could be recovered from small numbers of fixed cells, showing that GFP could be used as a convenient screenable marker for FACS separations. GFP was fused to the C-terminus of the LPG1 protein, which retained its ability to restore LPG expression when expressed in the lpg- R2D2 mutant of L. donovani. The LPG1(GFP) fusion was localized to a region situated between the nucleus and kinetoplast; its pattern was similar to that of LPG2, which is known to be located in the Golgi apparatus. This is notable as LPG1 participates in the biosynthesis of the glycan core of the LPG GPI anchor, whereas protein GPI anchor biosynthesis occurs in the endoplasmic reticulum. These studies suggest that the GFP will be a broadly useful marker in Leishmania.

More than 2 years have passed since legislation was first proposed in New York to remedy the absence of legal authority for family members who must make important health care decisions for incapacitated loved ones who have left no advance directives. This legislation, the "Family Health Care Decisions Act," included standards for surrogate decision-making and safeguards for surrogate decisions about the use of life-sustaining treatments. This article argues in favor of the legislation and discusses the concerns presented in opposition.

Various experiments have demonstrated a role for the E2F transcription factor in the regulation of cell growth during the G0/G1/S phase transition. Indeed, overexpression of the E2F1 product, a component of the cellular E2F activity, induces DNA synthesis in quiescent fibroblasts. To provide an approach to a more detailed biochemical analysis of these events, we have made use of a recombinant adenovirus containing the E2F1 cDNA in order to efficiently express the E2F1 product in an entire population of cells. We demonstrate an induction of DNA synthesis when quiescent cells are infected with the E2F1 recombinant virus. However, we also find that the induction does not lead to a complete replication of the cellular genome, as revealed by flow cytometry. The incomplete nature of cellular DNA replication is due, at least in part, to the fact that E2F1 overexpression leads to massive cell death that is characteristic of apoptosis. This E2F1-mediated induction of apoptosis is largely dependent on endogenous wild-type p53 activity and can be subverted by introducing mutant forms of p53 into these cells or by overexpressing E2F1 in fibroblasts derived from p53-null mouse embryos. We conclude that E2F1 can induce events leading to S phase but that the process is not normal and appears to result from the activation of a cell death pathway.

Ceramide, a lipid mediator, has been most closely associated with antiproliferative activities. In this study, we examine the mechanism by which ceramide induces growth suppression and the role of the retinoblastoma gene product (Rb) in this process. Withdrawal of serum from the serum-dependent MOLT-4 cells resulted in significant dephosphorylation of Rb, correlating with the induction of G0/G1 cell cycle arrest. Serum withdrawal resulted in marked elevation in the levels of endogenous ceramide (3-fold at 24 h and 8-fold at 96 h) with little changes in the endogenous levels of sphingosine. The addition of exogenous C6-ceramide resulted in a concentration- and time-dependent dephosphorylation of Rb. Exogenous ceramide was active at levels comparable to endogenous levels achieved with serum withdrawal. Peak activity of exogenous ceramide (at 6 h) correlated with the uptake of C6-ceramide by MOLT-4 cells. Next, a number of studies were conducted to determine whether Rb plays a role in ceramide-induced growth suppression. (i) C6-Ceramide was poorly active in growth suppression of retinoblastoma cells that lack Rb. (ii) Mink lung epithelial cells in which Rb had been sequestered by overexpression of large tumor antigen were resistant to the action of ceramide compared to cells transfected with large tumor antigen mutated in the Rb-binding pocket. (iii) Overexpression of the EIA adenoviral protein, which binds and sequesters Rb, resulted in protection from growth suppression and cell cycle arrest induced by ceramide. Thus, these studies demonstrate that Rb is a downstream target for ceramide and may function in a growth suppressor pathway resulting in cell cycle arrest.

Inhibition of cell growth by type beta transforming growth factor (TGF-beta) occurs in mid-G1 and is associated with decreased G1 cyclin-dependent kinase activity and maintenance of the retinoblastoma tumor suppressor protein Rb in an underphosphorylated, growth-suppressive state. A variety of recent experiments suggest that a functional target of Rb is the E2F transcription factor. In addition, the growth-suppressive effects of TGF-beta can be overcome by expression of viral oncogene products that dissociate E2F from Rb and Rb-related polypeptides. These results suggest the possibility that control of E2F may be a downstream event of TGF-beta action. Consistent with that possibility is the observation that E2F1 RNA levels are drastically reduced in TGF-beta-treated cells. We have also used a recombinant adenovirus containing the human E2F1 gene to overexpress the E2F1 product in mink lung epithelial cells that were growth arrested with TGF-beta. We find that overexpression of E2F1 can overcome the TGF-beta-mediated effect as measured by the activation of cellular DNA synthesis. These results suggest that a likely downstream target for the cyclin-dependent kinases, which are controlled by TGF-beta, is the activation of E2F.

Several lines of evidence implicate the E2F transcription factor as an important component of cell proliferation control. First, E2F binding sites are found in the promoters of genes responsive to proliferation signals and the level of E2F binding activity increases at a time when many of these genes are activated. Second, the tumour suppressor protein Rb, as well as the related p107 protein, complexes with E2F, resulting in an inhibition of E2F transcriptional activity. Third, oncogenic products of the DNA tumour viruses can dissociate these E2F complexes. We provide here direct evidence that E2F is involved in cellular proliferation control. Specifically, we demonstrate that overexpression of the E2F1 complementary DNA can activate DNA synthesis in cells that would otherwise growth-arrest, with an efficiency that is similar to that achieved by the expression of the adenovirus E1A gene. Moreover, microinjection of the E2F1 cDNA into quiescent cells can induce S-phase entry, whereas two E2F1 mutants, which are unable to transactivate the DHFR and TK promoters, are unable to induce S phase. We conclude that the E2F transcription factor plays an important role in progression into S phase and that this probably coincides with its capacity to stimulate transcription.

"When Others Must Choose: Deciding for Patients Without Capacity" is health care legislation proposed for New York state. Its intent is to remedy the absence of legal authority for families or other surrogates to make treatment decisions for their incapacitated loved ones who have neither completed an advance directive nor designated a health care agent. This paper provides a summary of this legislation and highlights nursing implications and the role of the nurse as a member of institutional ethics committees.

The E2F transcription factor is found in complexes with a variety of cellular proteins including the retinoblastoma tumor suppressor protein. Various assays have demonstrated a tight correlation between the functional capacity of Rb as a growth suppressor and its ability to bind to E2F. Moreover, only the underphosphorylated form of Rb, which appears to be the active species, interacts with E2F. Despite the fact that the majority of Rb becomes hyperphosphorylated at the end of G1, we now show that the E2F-Rb interaction persists through the G1/S transition and into S phase. A distinct E2F complex does appear to be regulated in relation to the transition from G1 to S phase. We now demonstrate that this complex contains the Rb-related p107 protein. Moreover, like the Rb protein, p107 inhibits E2F-dependent transcription in a co-transfection assay. This result, together with the observation that free, uncomplexed E2F accumulates as cells leave G1 and enter S phase, suggests that the p107 protein may regulate E2F-dependent transcription during G1. In contrast, although Rb does regulate the transcriptional activity of E2F, this association does not coincide with the G1 to S phase transition.

Schwarz discusses the uses and limitations of advance written directives and health care proxies as means to patient self-determination in health care decision making, as well as implications for nursing practice.

The translocation of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) across rat liver Golgi-derived vesicles has been studied. Vesicles of the same topographical orientation as in vivo were incubated with a mixture of [adenine-8-3H]PAPS and [35S]PAPS. The tritium to radiolabeled sulfur ratio of the incubation medium was 1.73 +/- 0.03 while that in the vesicles was 1.82 +/- 0.13. This strongly suggests that the entire PAPS molecule was being translocated across the Golgi vesicle membrane even though intact PAPS could not be detected within the vesicles. Translocation of PAPS resulted in accumulation of solutes within vesicles. This accumulation was temperature dependent, saturable (apparent Km = 0.7 microM; Vmax = 25 pmol/mg of protein/10 min), and inhibited by the substrate analogue 3',5'-ADP but not by 2',5'-ADP. Translocation of PAPS was inhibited following treatment of Golgi vesicles with Pronase under conditions in which the activity of a lumenal Golgi membrane marker such as sialyltransferase was not. This result is consistent with the existence of a PAPS carrier protein, portions of which face the cytoplasmic side of the Golgi membrane.