Minimal/underreported but definite risk of death/bodily harm threats (DBHTs) to pain practitioners: results of nationwide survey from United States.

BACKGROUND: Many physicians have experienced or will experience patient who acts threateningly towards them at least once in their careers. However, there have been no studies to gauge the incidence rate and severity of patients' and/or patients' families' violence towards pain physicians. OBJECTIVES: This nationwide survey was completed to evaluate the incidence of death/bodily harm threats (DBHTs) against pain physicians. METHODS: A questionnaire along with online assent form was uploaded on SurveyMonkey Online Portal. The uploaded survey web-link was sent to pain fellowship programs in the United States so that pain physicians and pain fellows can respond to this survey. The respondents were expected to anonymously complete the survey containing various questions relating to confrontational patients' experiences, how these experiences affected them, how those situations were handled, and how the respondents would act differently in the future secondary to their victimization by the confrontational patients. RESULTS: The response rate to the nationwide survey was extremely low (5.2% of anticipated numbers), most likely secondary to underreporting. Out of total 26 respondents across the United States, seven respondents reported receiving DBHTs (incidence of 27%). The median number of absolute DBHTs received in lifetime by these seven respondents was three (range being 1 to 21-30). CONCLUSION: There is minimal/underreported but definite risk of DBHTs for pain practitioners and the improved reporting, awareness and discussions can help pain physician community to formulate efficacious strategies to the prevention and management of future DBHTs.

Nicotinic acetylcholine receptor gene expression is altered in burn patients.

INTRODUCTION: Burn patients have been observed to be more susceptible to the hyperkalemic effect of the depolarizing muscle relaxant succinylcholine. Changes in nicotinic acetylcholine receptor (nAChR) subunit composition may alter electrophysiologic, pharmacologic, and metabolic characteristics of the receptor inducing hyperkalemia on exposure to succinylcholine. No studies have been performed that show the upregulation and/or alteration of nAChR subunit composition in human burn patients. The scarcity of studies performed on humans with burn injury is mainly attributable to the technical and ethical difficulties in obtaining muscle biopsies at different time frames of illness in these acutely injured patients. nAChRs are expressed in oral keratinocytes and are upregulated or altered in smokers. However, no studies have addressed the expression of nAChRs in the oral mucosa of burn patients. METHODS: Buccal mucosal scrapings were collected from 9 burn patients and 6 control nonburn surgical intensive care unit patients. For burn and control patients, tissues were collected upon presentation (time: 0 hour) and at time points 12, 24, and 48 hours, 1 week, and 2 weeks. Gene expression of the nAChR subunits alpha1, alpha7, gamma, and epsilon were performed using real-time reverse transcriptase polymerase chain reaction. RESULTS: alpha7 and gamma nAChR genes were significantly upregulated in burn patients, whereas alpha1 and epsilon nAChR genes were minimally affected, showing no significant changes over time. DISCUSSION: Over the 2 weeks of measurement, an upregulation of the alpha7 and gamma genes occurred in both burn and control patients; however, the proportion of alpha7 and gamma subunit increases was significantly higher in burn patients than in control surgical intensive care unit patients. The relationship between the thermal injury and the observed alteration in gene expression suggests a possible cause/effect relationship. This effect was observed at a site not affected by the burn injury and in nonmuscle tissues, thus emphasizing the systemic nature of the effect caused by the thermal injury. Because gene expression is the basis of protein production, the upregulation of alpha7 and gamma genes might translate into more alpha7 and gamma protein subunits. These proteins can also combine with each other or with other types of subunits (alpha1, beta, epsilon . . .) to form nAChRs with altered electrophysiologic characteristics leading to the observed abnormal clinical outcomes. CONCLUSION: Thermal injury may infer a systemic effect because upregulation/alteration of nAChRs occurs in nonmuscle tissues distant from the site of injury. The effect of thermal injury on nAChR gene subunits can be studied using a minimally invasive method (buccal mucosal scraping) and a highly sensitive technology (real-time reverse transcriptase polymerase chain reaction) obviating the need for more invasive methods.

Sphingosine kinase 1 is up-regulated in colon carcinogenesis.

Sphingosine kinase 1 (SK1) phosphorylates sphingosine to form sphingosine 1-phosphate (S1P), which has the ability to promote cell proliferation and survival and stimulate angiogenesis. The SK1/S1P pathway also plays a critical role in regulation of cyclooxygenase-2 (COX-2), a well-established pathogenic factor in colon carcinogenesis. Therefore, we examined the expression of SK1 and COX-2 in rat colon tumors induced by azoxymethane (AOM) and the relationship of these two proteins in normal and malignant intestinal epithelial cells. Strongly positive SK1 staining was found in 21/28 (75%) of rat colon adenocarcinomas induced by AOM, whereas no positive SK1 staining was observed in normal mucosa. The increase in SK1 and COX-2 expression in AOM-induced rat colon adenocarcinoma was confirmed at the level of mRNA by real-time RT-PCR. In addition, it was found that 1) down-regulation of SK1 in HT-29 human colon cancer cells by small interfering RNA (siRNA) decreases COX-2 expression and PGE2 production; 2) overexpression of SK1 in RIE-1 rat intestinal epithelial cells induces COX-2 expression; and 3) S1P stimulates COX-2 expression and PGE2 production in HT-29 cells. These results suggest that the SK1/S1P pathway may play an important role in colon carcinogenesis, in part, by regulating COX-2 expression and PGE2 production.

EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy.

EpCAM (epithelial cell adhesion molecule) is a cell surface molecule that is known to be highly expressed in colon and other epithelial carcinomas. EpCAM is involved in cell-to-cell adhesion and has been the target of antibody therapy in several clinical trials. To assess the value of EpCAM as a novel target for breast cancer gene therapy, we performed real-time reverse transcription-PCR to quantify the level of EpCAM mRNA expression in normal breast tissue and primary and metastatic breast cancers. We found that EpCAM is overexpressed 100- to 1000-fold in primary and metastatic breast cancer. Silencing EpCAM gene expression with EpCAM short interfering RNA (siRNA) resulted in a 35-80% decrease in the rate of cell proliferation in four different breast cancer cell lines. EpCAM siRNA treatment decreased cell migration by 91.8% and cell invasion by 96.4% in the breast cancer cell line MDA-MB-231 in vitro. EpCAM siRNA treatment was also associated with an increase in the detergent-insoluble protein fraction of E-cadherin, alpha-catenin, and beta-catenin, consistent with the known biology of EpCAM as a regulator of cell adhesion. Our hypothesis is that modulation of EpCAM expression can affect cell migration, invasion, and proliferation by enhancing E-cadherin-mediated cell-to-cell adhesion. These data provide compelling evidence that EpCAM is a potential novel target for breast cancer gene therapy and offer insights into the mechanisms associated with EpCAM gene silencing.

Molecular detection of breast cancer cells in the peripheral blood of advanced-stage breast cancer patients using multimarker real-time reverse transcription-polymerase chain reaction and a novel porous barrier density gradient centrifugation technology.

PURPOSE: The goal of this study was to develop a molecular diagnostic assay to detect circulating breast cancer cells in the peripheral blood for the purpose of staging breast cancer. Our aim was to make available an assay that was not limited by the low concentration of circulating breast cancer cells and the background gene expression that is typically found in peripheral blood. EXPERIMENTAL DESIGN: In this study, we investigated the ability of two new technologies to significantly enhance the quantification of gene expression in the peripheral blood: enrichment by a novel porous barrier density gradient centrifugation technology; and multimarker real-time reverse transcription-PCR (RT-PCR). RESULTS: Using fluorescence-labeled breast cancer cells and flow cytometry, we show that processing peripheral blood by porous barrier density gradient centrifugation results in a 300-fold enrichment of breast cancer cells. Real-time RT-PCR analysis confirmed a concomitant reduction in background expression of the CK19 and MUC1 genes after enrichment. In a pilot study, porous barrier density gradient centrifugation and multimarker real-time RT-PCR enabled our laboratory to detect breast cancer-associated gene overexpression in 13 of 20 (65%) stage IV breast cancer patients. Nine of these 14 patients overexpressed three or more markers. CONCLUSIONS: These results confirm the promise of such a molecular diagnostic assay and suggest that additional studies are needed to precisely define the clinical relevance.

Role for mammalian neutral sphingomyelinase 2 in confluence-induced growth arrest of MCF7 cells.

Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth (Marchesini, N., Luberto, C., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 13775-13783). In this study, the role of endogenous nSMase2 in regulating growth arrest was investigated. The results show that endogenous nSMase2 mRNA was up-regulated approximately 5-fold when MCF7 cells became growth-arrested at confluence, and total neutral SMase activity was increased by 119 +/- 41% with respect to control. Cell cycle analysis showed that up-regulation of endogenous nSMase2 correlated with G(0)/G(1) cell cycle arrest and an increase in total ceramide levels (2.4-fold). Analysis of ceramide species showed that confluence caused selective increases in very long chain ceramide C(24:1) (370 +/- 54%) and C(24:0) (266 +/- 81%) during arrest. The role of endogenous nSMase2 in growth regulation and ceramide metabolism was investigated using short interfering RNA (siRNA)-mediated loss-of-function analysis. Down-regulation of nSMase2 with specific siRNA increased the cell population of cells in S phase of the cell cycle by 59 +/- 14% and selectively reverted the effects of growth arrest on the increase in levels of very long chain ceramides. Mechanistically, confluence arrest also induced hypophosphorylation of the retinoblastoma protein (6-fold) and induction of p21(WAF1) (3-fold). Down-regulation of nSMase2 with siRNA largely prevented the dephosphorylation of the retinoblastoma protein and the induction of p21(WAF1), providing a link between the action of nSMase2 and key regulators of cell cycle progression. Moreover, studies on nSMase2 localization in MCF7 cells showed that nSMase2 distributed throughout the cells in subconfluent, proliferating cultures. In contrast, nSMase2 became nearly exclusively located at the plasma membrane in confluent, contact-inhibited cells. Hence, we demonstrate for the first time that nSMase2 functions as a growth suppressor in MCF7 cells, linking confluence to the G(0)/G(1) cell cycle check point.

Down-regulation of sphingosine kinase-1 by DNA damage: dependence on proteases and p53.

Sphingosine kinase 1 (SK1), a key enzyme in sphingosine 1-phosphate (S1P) synthesis, regulates various aspects of cell behavior, including cell survival and proliferation. DNA damaging anti-neoplastic agents have been shown to induce p53, ceramide levels, and apoptosis; however, the effects of anti-neoplastic agents on SK have not been assessed. In this study, we investigated the effects of a DNA damaging agent, actinomycin D (Act D), on the function of sphingosine kinase (SK1). Act D caused a reduction in the protein levels of SK1, as indicated by Western blot analysis, with a concomitant decrease in SK activity. The down-regulation was post-transcriptional, because the mRNA levels of SK1 remained unchanged. Similar decreases in SK1 protein were observed with other DNA damaging agents such as doxorubicin, etoposide, and gamma-irradiation. ZVAD, the pancaspase inhibitor, and Bcl-2 annulled the effect of Act D on SK1, demonstrating a role for cysteine proteases downstream of Bcl-2 in the down-regulation of SK1. Inhibition of caspases 3, 6, 7, and 9 only partially reversed Act D-induced SK1 loss. Inhibition of cathepsin B, a lysosomal protease, produced a significant reversal of SK1 decline by Act D, suggesting that a multitude of ZVAD-sensitive cysteine proteases downstream of Bcl-2 mediated the SK1 decrease. When p53 up-regulation after Act D treatment was inhibited, SK1 down-regulation was rescued, demonstrating p53 dependence of SK1 modulation. Treatment of cells with S1P, the product of SK1, partially inhibited Act D-induced cell death, raising the possibility that a decrease in SK1 may be in part necessary for cell death to occur. Furthermore, the knockdown of SK1 by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability. These studies demonstrate that SK1 is down-regulated by genotoxic stress, and that basal SK1 function may be necessary for the maintenance of tumor cell growth.

Defects in cell growth regulation by C18:0-ceramide and longevity assurance gene 1 in human head and neck squamous cell carcinomas.

In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/mass spectroscopy. Interestingly, only one specific ceramide, C(18:0)-ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues. On the other hand, in nonsquamous tumor tissues, this selectivity for C18-ceramide was not detected. These data suggested the hypotheses that decreased levels of C18-ceramide might impart a growth advantage to HNSCC cells and that increased generation of C18-ceramide may be involved in the inhibition of growth. These roles were examined by reconstitution of C18-ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C18-ceramide. Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/mUOG1 resulted in increased levels of only C(18:0)-ceramide by approximately 2-fold, i.e. concentrations similar to those of normal head and neck tissues. Importantly, increased generation of C18-ceramide by mLAG1/mUOG1 inhibited cell growth (approximately 70-80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction. In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C18-ceramide in the regulation of growth of HNSCC.