New Trends at 25 Years: An American Board of Surgery Academic Certifying Examination Review Course.

BACKGROUND: In the 1980s, a small research group began identifying variables affecting applicant success on the American Board of Surgery (ABS) Certifying Examination (CE). We now report success and trends as we complete 25 years. We had multiple challenges as identified through faculty focus groups and participant feedback that needed to be addressed: increase the national optics of the program, integrate new innovative experiences, maintain the integrity of the collected data on excel files, incorporate national trends in surgery, attract experienced clinical volunteer faculty and staff, security of capital, and schedule management. METHOD: The primary purpose of the program is to define the root cause interfering with success on the ABS CE. All of the listed changes in course design (2012-2016) were entered into excel files along with participants demographics, including results of the pretesting modules, the communication inventory, all self-reported stressors, and interview results to track the effect of faculty interventions, trends and ABS outcomes. RESULTS: The profiles of the participants have changed over time, including: marital status, presence of DSM-5 stressors, gender, fellowship training, study habits, financial burdens, and international graduate status. International graduates demonstrated communication issues that were present, though rarely addressed, during residency training. The gradual absorption of junior faculty allowed a seamless transition over time as part of the succession plan. Although the national success rates on the CE were 72% to 80%, this program's success rate still remained in the 90 percentile (94%-97%) for those who followed their education improvement plan. Deidentified excel files will be converted to REDCap for preservation and analysis. DISCUSSION: The small course design has continued to be effective at identifying variables that interfere with success on the CE examination. The inclusion of additional PhD education scientists facilitated focused individual interventions. A pilot program for international graduate status residents is in development.

The hottest sentinel lymph node is not always the positive node.

The technique of identifying the sentinel lymph node (SLN) varies from each individual institution. Generally, the highest isotope count in a lymph node is considered the SLN, whereas other radioactive nodes might also be removed. The purpose of our study was to determine if the hottest node was always the tumor-containing node. Two hundred forty-seven breast cancer patients underwent SLN biopsy from April 1998 to April 2002. Lymphatic mapping involved a radiocolloid injection and lymphoscintigraphy followed by intraoperative assessment with a hand-held gamma probe. All SLN(s) with radioactive counts 10 per cent or more of the ex vivo counts of the most radioactive SLN were removed. The SLN were sliced at 2-mm intervals with 4-microm step-sections (92-microm spacing) and evaluated by microscopy and immunohistochemistry. One hundred twenty (49%) of the 247 patients had 2 or more nodes resected. Of these 120 patients, 33 (28%) had a tumor-bearing node. In 25 (74%) cases, the tumor-bearing node was the most radioactive; however, in 8 (26%) cases, the positive node was a lesser reactive node. Although the most radioactive node in a draining basin is considered the SLN, this is often not the metastatic node. Therefore, all nodes with significant radioactive counts must be removed to ensure accurate staging.

Nuclear factor-kappa B is upregulated in colorectal cancer.

BACKGROUND: Chemoresistance may involve the anti-apoptotic transcriptional regulator, nuclear factor-kappa B (NF-kappa B). The purpose of this study was to determine whether chemotherapy induces NF-kappa B activation in a human colon cancer cell line (SW48) and whether NF-kappa B is constitutively activated in colorectal cancer. METHODS: SW48 cells were incubated with gemcitabine hydrochloride (Gemzar) in the presence and absence of the 26s proteasome inhibitor, MG132, and NF-kappa B binding (electrophoretic mobility shift assay), DNA synthesis (tritiated thymidine uptake), cell viability (3-[4,5-dimethylthiazol-2-yl]-diphenyl-tetrazolium bromide assay), and apoptosis (caspase-3 activity) were measured at 24 hours. NF-kappa B binding (electrophoretic mobility shift assay) was also assayed in 10 colorectal cancer tumors. RESULTS: SW48 cells demonstrated constitutive NF-kappa B binding that was enhanced by gemcitabine hydrochloride in a dose-dependent manner. MG132 inhibited NF-kappa B binding and enhanced gemcitabine hydrochloride's inhibition of DNA synthesis (gemcitabine hydrochloride = 73% +/- 1.4% vs gemcitabine hydrochloride + MG132 = 6% +/- 0.4%, P <.05), cell killing (gemcitabine hydrochloride = 87% +/- 2.0 vs gemcitabine hydrochloride + MG132 = 25% +/- 1.3%, P <.05), and caspase-3 activity (gemcitabine hydrochloride = 870 +/- 17.4 vs gemcitabine hydrochloride + MG132 = 1075 +/- 20.4, P <.05). NF-kappa B binding was increased in 8 of 10 colorectal cancer tumors compared with adjacent normal mucosa. CONCLUSIONS: Gemcitabine hydrochloride enhances NF-kappa B binding in a colorectal cancer cell line, whereas inhibition of NF-kappa B enhances gemcitabine hydrochloride's antitumor activity. NF-kappa B is also activated in human colorectal cancer. NF-kappa B may identify chemoresistant tumors, whereas inhibition of NF-kappa B may be a novel, biologically based therapy. (Surgery 2001;130:363-9).

Institutional validation of breast cancer treatment guidelines.

Several groups have developed clinical guidelines for the management of breast cancer, yet little data exist regarding their validation. Therefore, we examined the effect of published National Comprehensive Cancer Network (NCCN) guidelines for invasive breast cancer on survival, quality of life (QOL), and hospital cost. From 260 consecutive breast cancer patients, 129 patients were identified for analysis: 93 patients (72%) were treated according to the guidelines (NCCN+), while the treatment of 36 patients (28%), with a similar stage distribution, deviated from the guidelines (NCCN-). Patients were excluded from analysis with a diagnosis of carcinoma in situ, inflammatory cancer, stage IV disease, and comorbid conditions that affected treatment. The 5-year survival was 87.6% for the NCCN+ patients versus 83.3% for NCCN- patients (P = 0.319 by Kaplan-Meier). Twelve QOL parameters were evaluated using a Likert-type scale (1 = severe and 5 = none). NCCN+ patients had a cumulative QOL score of 4.18 +/- 0.08 versus 4.24 +/- 0.14 for NCCN- patients (P = 0.745). Treatment-related costs were $20,300 +/- 1800 for NCCN+ patients versus $59,700 +/- 25,200 for NCCN- patients (P = 0.016 by t test). Although deviation from NCCN breast cancer guidelines had no effect on perceived quality of life or survival, there was a significant decrease in cost in the NCCN+ group. These findings suggest that adherence to NCCN guidelines can significantly reduce the cost of breast cancer care without adversely affecting either survival or quality of life.

Local excision and postoperative radiation therapy for rectal adenocarcinoma.

Sixty-seven patients with early-stage adenocarcinoma of the rectum who had lesions thought to be unsuitable for either local excision alone or endocavitary irradiation were treated with local excision followed by postoperative radiation therapy. The purpose of this study was to evaluate the effectiveness of local excision followed by radiation therapy for treatment of rectal adenocarcinoma. The patients were treated between 1974 and 1999; follow-up time was 6 to 273 months (median, 65 months). All living patients had follow-up for at least 2 years. The indications for postoperative irradiation included equivocal or positive margins, invasion of the muscularis propria, endothelial-lined space invasion, poorly differentiated histology, and perineural invasion. Cox proportional hazards regression analysis was performed using six explanatory variables including tumor size, configuration (exophytic vs. ulcerative), histologic differentiation, pathologic T stage, endothelial-lined space invasion, and margin status. The time interval between treatment and development of recurrent disease was in the range of 11 to 48 months. The 5-year results were as follows: local-regional control, 86%; ultimate local-regional control, 93%; distant metastasis-free survival, 93%; absolute survival, 80%; and cause-specific survival, 90%. When the Cox proportional hazards regression analysis was performed for these endpoints, margin status influenced absolute survival (P = 0.0074), cause-specific survival (P = 0.0405), and ultimate local-regional control (P = 0.0439). Tumor configuration marginally influenced cause-specific survival (P = 0.0577). None of the variables had an influence on the endpoints' local-regional control, ultimate local-regional control with sphincter preservation, or distant metastasis. Five patients (7%) had severe complications; no complication was fatal. Local excision and postoperative radiation therapy results in a high probability of local-regional control and survival for selected patients with relatively early-stage rectal adenocarcinoma. Patients with ulcerative tumors may have a lower likelihood of cause-specific survival.

The effect of the duration and structure of a surgery clerkship on student performance.

The emphasis on a generalist professional education has led to shortening and restructuring of the surgery clerkship in the curricula of many medical schools. Little data exist regarding the effect of these changes on student performance. Therefore, we examined the effect of the length, timing, and content of the third year surgery rotation on several clerkship and postclerkship performance measures of 487 students from July 1994 to July 1998. In addition, students' perceptions regarding their ability to understand surgical disease topics were surveyed. The 8-week clerkship (n = 232) was associated with higher NMBE surgery test scores (510.5 +/- 6.3 versus 457.4 +/- 10.0, P < 0.05) resulting in higher final clerkship grades (5.15 +/- 0.04 versus 4.87 +/- 0.03, P < 0.05). Although clerkship length had no significant effect on USMLE step 2 total or surgery subsection scores, the longer clerkship was associated with higher total (70.6 +/- 0.37 versus 68. 8 +/- 0.50, P < 0.05) and abdominal pain station (81.87 +/- 0.71 versus 79.54 +/- 0.73, P < 0.05) OCSE scores. Students rotating on surgery during the second half of third year (n = 233) had higher NMBE surgery test scores (513.1 +/- 8.9 versus 460.5 +/- 11.2, P < 0. 05) and final grades (5.17 +/- 0.03 versus 4.81 +/- 0.04, P < 0.05). Although the timing of the surgery clerkship did not significantly affect total OSCE scores, students who rotated on surgery in the second half of third year performed significantly better year on the abdominal pain OSCE station (80.47 +/- 0.92 versus 76.49 +/- 1.27, P < 0.05). Students who rotated on general surgery (n = 298) performed significantly better on the NBME surgery test (525.6 +/- 6.0 versus 459.6 +/- 9.1, P < 0.05), although this did not significantly affect the final grade. Although general versus subspecialty surgery rotation did not significantly affect total OSCE scores, students rotating on general surgery performed significantly better on the abdominal pain OSCE station (81.21 +/- 0.91 versus 78.17 +/- 0.32, P < 0.05). The length, timing, and content of the third year surgery rotation had no significant effect on performance on the oral examination. Students who had a 6-week clerkship and students who lacked exposure to general surgery felt their surgery rotation failed to prepare them to understand a number of surgical topics as well as students who had an 8-week clerkship or students who rotated on general surgery. The length, timing, and content of the surgery clerkship affect some clerkship performance measures and student perceptions of their understanding of surgical disease topics. While cognitive differences related to clerkship length are no longer detectable at the end of the third year of medical school, differences related to the acquisition of some clinical skills persist after the surgery clerkship.

Stereotactic core biopsy reduces the reexcision rate and the cost of mammographically detected cancer.

The management of patients with mammographic abnormalities is rapidly shifting from needle-localized surgical biopsy (NLB) to stereotactic core biopsy (SCB). The precise role of SCB in the management of nonpalpable breast cancer remains to be defined. The purpose of this study was to compare SCB to NLB in the diagnosis of mammographically detected breast cancer in women who underwent breast-conserving surgery. The records of all patients with nonpalpable breast cancer who underwent breast-conserving surgery from 1/1/95 to 6/1/97 were analyzed with respect to method of diagnosis, time interval from detection to diagnosis and breast-conserving surgery, volume of breast tissue excised, margin status and reexcision rate, number of surgical procedures, and total charges and costs per patient. During a 30-month period, 117 patients with nonpalpable breast cancer underwent breast-conserving surgery. The diagnosis was made by NLB in 69 patients and SCB in 48 patients. The time from detection to diagnosis and breast-conserving surgery was 1.7 +/- 0.5 and 8.1 +/- 1.2 days for SCB patients and 6. 8 +/- 1.3 and 16.9 +/- 2.3 days for NLB patients (P < 0.01). The volume of breast tissue removed was 117.9 +/- 5.6 cm3 for SCB patients versus 75.2 +/- 2.9 cm3 for NLB patients (P < 0.01). Three SCB patients (6%) had positive margins, while 38 NLB patients (55%) had positive margins (P < 0.01). Only 1 SCB patient (2%) was reexcised, while 34 NLB patients (50%) were reexcised (P < 0.01). Eighty-nine percent of SCB patients had a single surgical procedure compared to 39% of NLB patients (P < 0.001). Patients who underwent SCB had reduced total charges and total costs per patient compared to NLB patients ($11,700 +/- $554 and $3537 +/- $167 per SCB patient versus $15,654 +/- $706 and $4853 +/- $198 per NLB patient, P < 0. 0001). Stereotactic core biopsy shortens the time from detection at mammography to diagnosis and breast-conserving therapy, permits appropriate discussion of treatment alternatives, reduces the positive margin rate and reexcision rate, and may represent a significant cost savings in the management of nonpalpable breast cancer.

Arginine-enhanced enteral nutrition augments the growth of a nitric oxide-producing tumor.

BACKGROUND: Arginine-enhanced diets have been shown to be beneficial in tumor-bearing hosts, but no data exist regarding their effects in hosts bearing nitric oxide (NO)-producting tumors. OBJECTIVE: To examine the effect of arginine supplementation on the growth of a NO-producing murine breast cancer cell line. METHODS: EMT-6 cells were grown in various concentrations of arginine in the presence or absence of the inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (1 mmol/L). Forty-eight hours later, nitrite accumulation and viable cell number were assessed. BALB/c mice were then pair-fed basal purified diets (n = 10), 4% casein diets (isonitrogenous control, n = 5), or 4% arginine-enhanced diets (n = 10). One week later, 10(5) EMT-6 cells were implanted subcutaneously into the dorsal flank. After tumor implantation, five mice fed basal purified diets and five mice fed arginine-enhanced diets also received aminoguanidine (100 mg/kg subcutaneously twice daily). Two weeks after tumor cell implantation, tumor size (mean diameter), animal weight, serum and tumor nitrite and nitrate levels were measured. RESULTS: There was minimal nitrite accumulation in arginine-free media, while increasing the arginine concentration increased nitrite levels. Viable cell number did not increase in arginine-free media, but increased nearly twofold in 100 and 1000 mumol/L arginine. In 5000 and 10,000 mumol/L arginine, the difference in viable cell number was not statistically different than that seen in arginine-free media, whereas the addition of aminoguanidine blocked nitrite accumulation and increased viable cell number at these arginine concentrations. Arginine-enhanced diets stimulated tumor growth in vivo more than twofold over tumor growth in mice fed isonitrogenous control or basal purified enteral diets. Mice fed arginine-enhanced diets also had increased serum nitrite and nitrate levels over mice fed basal purified enteral diets, whereas tumors from mice fed arginine-enhanced diets had nitrite and nitrate levels similar to mice fed basal purified enteral diets. Aminoguanidine blocked the increase in serum nitrite and nitrate, but failed to block the increased tumor growth in mice receiving the arginine-supplemented diets. CONCLUSIONS: Arginine concentration influences the growth of EMT-6 tumor cells in vitro and dietary arginine supplementation augments tumor growth in vivo. The mechanism of the growth modulation in vitro is NO-dependent whereas the enhanced tumor growth in vivo is NO-independent.

Nitric oxide contributes to adriamycin's antitumor effect.

UNLABELLED: Recently, several antitumor drugs have been shown to stimulate nitric oxide (NO) production. PURPOSE: To determine if adriamycin induces NO production in breast cancer cells in vitro and whether NO contributes to adriamycin's antitumor effect in vivo. METHODS: Murine breast cancer cells (EMT-6) were incubated with adriamycin (ADRIA, 0, 10, 100, 1000 microM) in the presence or absence of the NO synthase inhibitor aminoguanidine (AG, 1 mM). Twenty-four hours later nitrite accumulation (Greiss reagent) and cell viability (MTT assay) were assessed. Supernatants from adriamycin-stimulated cells were also analyzed at 6, 8, and 24 hr for TNF, IL-1, and IFN gamma (ELISA). For in vivo experiments, 10(5) EMT-6 cells were injected into the flank of BALB/c mice (n = 20) and 1 hr later mice received one of four treatments: (1) saline, (2) ADRIA (10 mg/kg ip), (3) AG (100 mg/kg sc BID), or (4) ADRIA (10 mg/kg ip) and AG (100 mg/kg sc BID). Two weeks later tumor size was measured and in situ tumor cell apoptosis was determined by fluorescent microscopy and flow cytometry. RESULTS: Adriamycin was cytotoxic to EMT-6 cells with 100 microM resulting in nearly 100% killing (P < 0.01). Adriamycin also stimulated nitrite accumulation with 100 microM producing 6.5 +/- 0.26 microM nitrite (P < 0.001). AG blocked adriamycin-stimulated nitrite accumulation (P < 0.05), but did not inhibit cytotoxicity in vitro. In vivo, adriamycin inhibited tumor size by nearly 400% (P < 0.001), while AG attenuated adriamycin's effect on tumor growth (P < 0.05). There was no difference in the detection of apoptotic tumor cells between the adriamycin and adriamycin and AG groups as determined by immunohistochemistry and flow cytometry. CONCLUSIONS: These findings suggest that adriamycin stimulated NO production in EMT-6 cells, but adriamycin's cytotoxicity in vitro was NO-independent. In vivo, adriamycin inhibited tumorigenesis partially via an NO-dependent, nonapoptotic mechanism.

Increased L-arginine transport in a nitric oxide-producing metastatic colon cancer cell line.

BACKGROUND: Little is known about amino acid transport in human neoplastic cells. We previously characterized L-arginine transport in the primary human colon cancer cell line, SW480, and found it is principally mediated by the sodium-independent system y+. In this study, we characterized L-arginine transport in the metastatic cell line, SW620, and compared it with that in the primary cell line, SW480. METHODS: Transport of 3H-L-arginine in cell monolayers was analyzed in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transporter affinity (Km) and maximal transport velocity (Vmax). Transport was further characterized through blockade with known amino acids. In addition, the effect of cell age (i.e., time in culture) on arginine transport was examined at 2 and 9 days after seeding. Cellular proliferation was assessed by using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. RESULTS: L-Arginine uptake was primarily sodium independent in the SW620 cell line. Kinetic and amino acid-inhibition studies revealed a single high-affinity, sodium-independent L-arginine transporter (Vmax = 1286.3 +/- 158.3 pmol/mg protein/30 s; Km = 46.8 +/- 4.2 microM). Sodium-independent transport was blocked by system y+ substrates L-homoarginine, L-ornithine and L-lysine. Sodium-dependent uptake occurs through a single transporter with system BO,+ characteristics (Km = 16.15 +/- 2.1 microM; Vmax = 329.94 +/- 29.7 pmol/mg protein/30 s). Arginine transport increased with time in culture with day 2 cells transport velocity = 241.7 +/- 33.6 pmol/mg protein/30s, whereas day 9 cells transport velocity = 377 +/- 15.4 pmol/mg protein/30 s (p < 0.01). Cellular-proliferation studies revealed a doubling time of 3.2 days for SW620 and 5.4 days for SW480 (p < 0.05). CONCLUSIONS: L-Arginine transport in these neoplastic cell lines occurs primarily through sodium-independent, high-affinity system y+. Vmax was increased 180% in the metastatic variant (SW620), suggesting upregulation of the Y+ transporter. The increased Y+ activity may be a mechanism to provide continuous substrate for tumor growth.

Tumor cell nitric oxide inhibits cell growth in vitro, but stimulates tumorigenesis and experimental lung metastasis in vivo.

Arginine-derived nitric oxide (NO) has been identified in some tumor cell lines and solid human tumors. The effect of tumor cell NO on tumor biology is poorly understood. The purpose of this study was to investigate the effect of NO production by EMT-6 murine breast cancer cells on tumor cell growth in vitro and subcutaneous tumor growth and experimental pulmonary metastasis in vivo. EMT-6 cells were incubated with endotoxin (LPS, 10 microgram/ml) and interferon-gamma (IFN, 50 U/ml), in the presence or absence of the NO synthase inhibitor, omega-nitro-L-arginine methyl ester (L-NAME, 2 mM), and NO production and cell number were assessed 24 hr later. EMT-6 cells were also treated overnight with LPS/IFN, in the presence or absence of L-NAME, washed and injected either subcutaneously in the dorsal flank (n = 40) or via the tail vein (n = 40) of syngeneic BALB/c mice. Two weeks following tumor cell injection, tumor size and number of pulmonary metastases were assessed. LPS/IFN stimulated NO production in EMT-6 cells and inhibited cell growth in vitro by 50%. L-NAME blocked LPS/IFN stimulation of NO production and restored cell growth to near control levels. When injected into BALB/c mice, LPS/IFN-stimulated tumor cells demonstrated a two-fold increase in subcutaneous tumor growth and experimental pulmonary metastases over control cells. L-NAME reduced tumor size and number of lung metastases to control levels, suggesting that tumor cell NO production was responsible for this effect. In summary, LPS/IFN-stimulated NO production in EMT-6 tumor cells inhibits tumor cell growth in vitro, yet paradoxically augments tumor growth and metastasis in vivo.

Dietary modulation of amino acid transport in rat and human liver.

Specialized diets enriched in the amino acids glutamine and arginine have been shown to benefit surgical patients. In the liver, glutamine supports glutathione biosynthesis, arginine regulates nitric oxide synthesis, and both of these amino acids serve as precursors for ureagenesis, gluconeogenesis, and acute phase protein synthesis. The effects of a diet enriched with glutamine and arginine on hepatic plasma membrane transport activity have not been studied in humans. We hypothesized that feeding supradietary amounts of these nutrients would enhance the activities of the specific carriers which mediate their transmembrane transport in the liver. We fed surgical patients (n = 8) and rats (n = 6) one of three diets: a) a regular diet, b) an enteral liquid diet containing arginine and glutamine, or c) an enteral diet supplemented with pharmacologic amounts of glutamine and arginine. Diets were isocaloric and were administered for 3 days. Hepatic plasma membrane vesicles were prepared from rat liver and from human wedge biopsies obtained at laparotomy. The transport of glutamine and arginine by rat and human vesicles was assayed. Vesicle integrity and functionality were verified by osmolarity plots, enzyme marker enrichments, and time courses. Provision of both a standard enteral liquid diet and one enriched with glutamine and arginine increased the activities of Systems N (glutamine) and y+ (arginine) in rat and human liver compared to a control diet. The diet supplemented with glutamine and arginine was the most effective in increasing transport activity. We conclude that the liver responds to diets enriched with specific amino acids by increasing membrane transport activity. This adaptive response provides essential precursors for hepatocytes which may enhance hepatic synthetic functions during catabolic states. This study provides insights into the mechanisms by which enteral nutrition regulates nutrient transport at the cellular level and may provide a biochemical rationale for the use of formulas which are enriched with conditionally essential nutrients.

Inflammatory mediators stimulate arginine transport and arginine-derived nitric oxide production in a murine breast cancer cell line.

Inflammatory mediators stimulate arginine-derived nitric oxide (NO) production in a variety of cells. The purpose of this study was to determine if the inflammatory mediators, endotoxin (LPS) and interferon gamma (IFN), stimulate arginine transport and nitric oxide production in a murine breast cancer cell line. We also investigated the effect of the nitric oxide synthase (NOS) inhibitors, omega-nitro-L-arginine methyl ester (LNAME) and aminoguanidine (AG), as well as the effect of varying the concentration of L-arginine in the cellular media, on arginine transport and NO production in these tumors cells. Confluent EMT-6 murine breast cancer cells were incubated with LPS (10 microgram/ml) and IFN (50 units/ml) in the presence or absence of the NOS inhibitors, L-NAME (2 mM) or AG (1 mM), and arginine transport (using L-[3H]arginine) and NO production (the stable end-product nitrite was assayed using the Greiss reagent) were measured at various time points. In addition, the effect of varying the concentration of L-arginine (0, 10, 100, 1000, 10,000 mM) in the cellular media on stimulated L-arginine transport and nitrite accumulation was assessed. Incubation of EMT-6 with LPS and IFN stimulated arginine transport approximately 70% over control levels at 12 hr and transport returned to basal levels at 24 hr. LPS/IFN-stimulated EMT-6 cells produced 25 microM nitrite at 24 hr and reached a plateau of 55 microM nitrite at 48 hr. The NO synthase inhibitors, L-NAME and AG, failed to inhibit basal and stimulated levels of arginine transport, but significantly inhibited nitrite accumulation, which was restored by 10 mM L-arginine. Finally, L-arginine was necessary in the media for nitrite accumulation by LPS/IFN-stimulated cells, with maximal accumulation at 1 mM L-arginine. In summary, LPS/IFN stimulate arginine transport and NO production in the EMT-6 breast cancer cell line. L-NAME and AG do not inhibit basal or stimulated arginine transport in this tumor cell line and extracellular L-arginine is required for NO synthesis in these cells. LPS/IFN stimulation of arginine transport may represent an adaptive response to provide increased substrate for enhanced tumor cell NO production.

Management of recurrent colorectal cancer: another look at carcinoembryonic antigen-detected recurrence.

There is currently controversy regarding the use of serum carcinoembryonic antigen (CEA) in the follow-up of colorectal cancer. This article reviews the most recent clinical data regarding CEA and discusses the rationales for its use in the follow-up of colorectal cancer. The patterns of colorectal cancer recurrence and their variable association with CEA elevation are presented. The contributions of the new imaging modalities, including radiolabeled antibodies, are reviewed and information on the prognosis following resection of recurrences is provided. A practical approach to colorectal cancer recurrences is suggested in two algorithms.

TNF-stimulated arginine transport by human vascular endothelium requires activation of protein kinase C.

OBJECTIVE: The authors determined the endothelial arginine transport mechanism and the potential role of a tumor necrosis factor (TNF)-alpha-mediated signal transduction pathway involving protein kinase C (PKC) in regulating this transport in cultured endothelial cells. SUMMARY BACKGROUND DATA: The vascular endothelium metabolizes arginine to generate nitric oxide (NO), and an increase in NO production can be stimulated by several cytokines. The mechanism(s) responsible for the accelerated arginine transport are poorly understood. METHODS: Arginine transport was assayed in confluent human umbilical vein endothelial cells in the presence of TNF +/- the PKC inhibitor chelerythrine chloride. RESULTS: Carrier-mediated arginine transport was accomplished by two Na(+)-independent transporters, System y+ (80% of total transport) and System b0,+ (20% of transport). Tumor necrosis factor (0.1-2 ng/mL) increased System y(+)-mediated arginine transport in a time- and dose-dependent manner by augmenting System y+ transport maximal capacity (control Vmax = 1325 +/- 60 pmol/mg protein/minute vs. TNF Vmax = 3015 +/- 110 pmol/mg protein/minute, p < 0.01) without affecting transporter affinity (control Km = 30 +/- 1.4 microM vs. 34 +/- 1.3 microM arginine, p = NS). Stimulation was maximal at the 8-hour time point and was inhibited by both actinomycin D and cycloheximide. In addition, inhibition of PKC with chelerythrine abrogated the TNF-augmented arginine transport. Similarly, incubation of cells with the direct PKC activator TPA (phorbol ester 12-myristate 13-acetate) stimulated System y(+)-mediated arginine transport nearly fivefold, secondary to an increase in transporter Vmax (TPA Vmax = 5349 +/- 310 pmol/mg protein/minute, p < 0.001 vs. control), with no change in Km. This TPA-induced stimulation of arginine transport also was blocked by chelerythrine CI, actinomycin D, and cycloheximide. Incubation of TNF-stimulated cells with two NO synthase inhibitors did not reduce transport activity, suggesting that the arginine transporter and the NO synthase enzyme may, in part, be independently regulated.

Characterization and growth factor stimulation of L-arginine transport in a human colon cancer cell line.

BACKGROUND: Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are potent mitogens that contribute to abnormal growth regulation in colon cancer. Growth factors have been shown to regulate transmembrane nutrient uptake as an adaptive response to support cellular proliferation. METHODS: The transport of L-arginine by the SW480 primary human colon adenocarcinoma cell line was characterized by assaying the uptake of [3H]L-arginine in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transport affinity (Km) and maximal transport velocity (Vmax). To further characterize the specific transporters, [3H]L-arginine uptake was measured in the presence of selected amino acids, hormones, and under conditions of varying external pH. To investigate the effects of EGF and TGF alpha, cells were incubated with increasing doses of growth factors (1, 10, 50 ng/ml) and L-arginine transport was measured at various time intervals (8, 12, 24 h). Proliferation was assessed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 3 days after growth factor stimulation. RESULTS: The majority of carrier-mediated L-arginine transport was via a sodium-independent process (65-70%), whereas the remainder was sodium-dependent (28-30%). Diffusion contributed a small amount to total L-arginine uptake (2%). Kinetic studies of arginine transport revealed a single high-affinity Na(+)-independent transporter with a Km = 55.8 +/- 5.8 microM and a Vmax = 710.6 +/- 87.3 pM/mg protein/30 s. Na(+)-independent arginine uptake was pH-insensitive and markedly inhibited by system y+ substrates L-homoarginine, L-lysine, and L-ornithine. A single Na(+)-dependent transporter with a Km = 19.8 +/- 2.3 microM and a Vmax = 159.1 +/- 8.9 pM/mg protein/30 s was identified. Na(+)-dependent arginine uptake was inhibited by system BO,+ substrates L-lysine, L-ornithine, L-leucine, L-cysteine, and L-glutamine, but not by 2-methylaminoisobutyric acid. In addition, Na(+)-dependent arginine uptake was pH- and hormone-insensitive. Incubation with EGF or TGF alpha had no effect on Na(+)-independent L-arginine uptake; however, Na(+)-dependent uptake was enhanced 60% by EGF (10 ng/ml, p < 0.05) and 100% by TGF alpha (10 ng/ml, p < 0.05), whereas cellular proliferation was increased 27% by EGF (10 ng/ml, p < 0.05) and 37% by TGF alpha (10 ng/ml, p < 0.01). CONCLUSIONS: L-arginine transport in the SW480 colon cancer cell line is principally mediated by the Na(+)-independent system y+ and to a lesser extent by the Na(+)-dependent system BO,+. Furthermore, EGF and TGF alpha preferentially stimulate L-arginine uptake via the Na(+)-dependent transporter, ostensibly to accommodate for the mitogenic stimulus.

Cytokines regulate endotoxin stimulation of endothelial cell arginine transport.

BACKGROUND: Endotoxin (lipopolysaccharide) stimulates transmembrane L-arginine transport in pulmonary artery endothelial cells (PAECs). The proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) mediate many of the pathophysiologic effects of endotoxemia and sepsis. Endothelial cells secrete TNF and IL-1 in response to endotoxin. We hypothesize that lipopolysaccharide stimulation of plasma membrane L-arginine transport is mediated via an autocrine cytokine loop involving TNF and IL-1. METHODS: Confluent porcine PAECs were incubated with various concentrations of lipopolysaccharide, TNF, or IL-1, and arginine uptake was determined by assaying the uptake of 3H-L-arginine in the presence or absence of Na+ at different time points. PAECs were then incubated with lipopolysaccharide or saline solution after pretreatment with either anti-TNF antibody or IL-1-receptor antagonist, and transport was measured 12 hours later. RESULTS: Lipopolysaccharide, IL-1, and TNF all increased both Na+-dependent and Na+-independent carrier-mediated L-arginine transport in a fashion that was both time and dose dependent. Maximal increases in stimulated arginine uptake occurred 8 hours after exposure to the cytokines and 12 hours after exposure to lipopolysaccharide. Pretreatment of endothelial cells with anti-TNF antibody blocked lipopolysaccharide stimulation of both Na+-independent and Na+-dependent transport by 100% and 90%, respectively. In addition, IL-1-receptor antagonist inhibited lipopolysaccharide stimulation of both Na+-independent and Na+-dependent transport by 65% and 85%, respectively. CONCLUSIONS: The marked increase in carrier-mediated L-arginine transport activity produced by lipopolysaccharide, IL-1, and TNF may represent an adaptive response by the pulmonary endothelium to support arginine-dependent biosynthetic pathways during sepsis. Furthermore, lipopolysaccharide stimulation of arginine transport is mediated in part through an autocrine mechanism involving IL-1 and TNF.

Normalization of tumor-induced increases in hepatic amino acid transport after surgical resection.

BACKGROUND: The liver of the host with cancer requires increased amounts of amino acids to support the synthesis of glucose and key defense proteins. To study the effect of the growing tumor on hepatic amino acid uptake, the authors measured hepatic transport activity in tumor-bearing rats and in rats at various times after tumor resection. METHODS: Fischer-344 rats were implanted subcutaneously with methylcholanthrene-induced fibrosarcoma cells (MCA sarcoma). When the tumors reached 10% of body weight, hepatic amino acid transport activity was assayed or the animals underwent surgical removal of the tumor. In animals that underwent tumor excision, livers were removed at 1, 3, or 5 days post-resection, and hepatic plasma membrane vesicles (HPMVs) were prepared. Nontumor-bearing pair-fed rats undergoing sham implantation or sham resection served as controls. System N (glutamine), System A (MeAIB), and System y+ (arginine) transport activity were assayed, which allowed the authors to compare differences in tumor-induced rates of transport and the influence of resection on transport activity. RESULTS: System A transport activity was unaltered by tumor growth. In contrast, the presence of the growing tumor increased arginine and glutamine uptake by the liver. Hepatic glutamine transport remained elevated for 5 days after tumor resection, although by postoperative day 5 there was a trend toward normalization. In contrast, arginine transport remained increased by twofold onpost-resection day 1 and had normalized by postoperative day 3. The enhanced arginine transport was a result of an increase in maximal transport velocity (Vmax) rather than a change in carrier affinity. CONCLUSIONS: Increases in hepatic amino acid transport normalize within several days of tumor resection, indicating a key role for the tumor in the induction of this response. The observation that hepatic glutamine transport activity remains augmented after tumor resection longer than any other transporter studied suggests a key role for this amino acid in overall hepatic nitrogen metabolism and may partially explain the persistent glutamine depletion that is characteristic of the tumor-bearing host.

Endotoxin-induced nitric oxide production in pulmonary artery endothelial cells is regulated by cytokines.

BACKGROUND: L-Arginine is the sole precursor of nitric oxide (NO). Bacterial lipopolysaccharide (endotoxin) (LPS) stimulates carrier-mediated L-arginine transport in porcine pulmonary artery endothelial cells (PAECs) through an autocrine pathway that involves interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha). OBJECTIVES: To determine if Escherichia coli LPS stimulates NO synthesis in PAECs and, if so, if LPS stimulation of NO production is also mediated by autocrine secretion of IL-1 alpha and TNF-alpha. DESIGN: Monolayers of PAECs were incubated with various concentrations of LPS, recombinant human TNF-alpha, or IL-1 alpha, and total nitrate-nitrite accumulation was measured at different time points with the Greiss reagent following cadmium reduction. Release of TNF-alpha and IL-1 alpha release by LPS-stimulated PAECs were measured using the WEHI (for TNF-alpha) and A375.S2 (for IL-1 alpha) bioassays. The PAECs were then incubated with saline solution or LPS in the presence or absence of either a polyclonal antibody to human TNF or IL-1 receptor antagonist, and nitrate-nitrite accumulation was measured at 48 hours. RESULTS: Production of NO by PAECs was increased 230% by LPS (1 microgram/mL), 350% by TNF-alpha (1000 U/mL), and 240% by IL-1 alpha (1000 U/mL) (P < .05 vs control). The LPS-stimulated NO production was inhibited by IL-1 receptor antagonist (100 micrograms/mL) or antibody to TNF (10 micrograms/mL) to control levels (P < .05 vs LPS; difference vs saline solution was not significant). The LPS-stimulated TNF-alpha secretion by PAECs and TNF-alpha activity were maximal at 6 hours (400 +/- 42 pg/mL). The IL-1 alpha activity was not detectable in LPS-stimulated PAECs by the A375.S2 bioassay. CONCLUSIONS: Endotoxin, TNF-alpha, and IL-1 alpha stimulated NO synthesis in PAECs. Endotoxin-stimulated NO synthesis through an autocrine pathway involving the cytokines TNF-alpha and IL-1 alpha.

Postoperative radiotherapy improves survival in squamous cell carcinoma of the hypopharynx.

BACKGROUND: Postoperative radiation is considered to be "standard of care" therapy for advanced, resectable squamous cell carcinoma of the head and neck. This approach has been supported by retrospective data but has not been validated in randomized clinical trials. PATIENTS AND METHODS: The present analysis examined the clinical course of 110 patients with squamous cell cancer of the hypopharynx treated with surgery alone (n = 65) and postoperative radiotherapy alone (n = 45) between 1966 and 1990. Staging of patients was performed using the 1988 American Joint Committee on Cancer criteria. Cox regression analyses identified clinical and pathologic factors that were significant for disease-free and overall survival. Crude and adjusted cancer-specific survival rates were calculated. RESULTS: The postoperative radiotherapy group presented with more advanced disease than the surgery alone group (stage III and IV combined, 96% versus 77%, P = 0.015). Crude 5-year cancer-specific survival probabilities were 43% for the postoperative therapy group and 27% for the surgery alone group (P = NS). Adjusted 5-year survival rates, correcting for differences in significant prognostic variables between groups, were 18% and 48%, respectively, for the surgery and postoperative radiotherapy groups (P = 0.029). CONCLUSIONS: The addition of postoperative radiotherapy was associated with improved disease-free and adjusted overall cancer-specific survival in patients with advanced hypopharyngeal squamous cancer. The potential survival benefit of postoperative radiotherapy should be addressed in a randomized clinical trial.