Effect of Portacaval Anastomosis on Glutamine Synthetase Protein and Gene Expression in Brain, Liver and Skeletal Muscle

The effects of chronic liver insufficiency resulting from end-to-side portacaval anastomosis (PCA) on glutamine synthetase (GS) activities, protein and gene expression were studied in brain, liver and skeletal muscle of male adult rats. Four weeks following PCA, activities of GS in cerebral cortex and cerebellum were reduced by 32% and 37% (p<0.05) respectively whereas GS activities in muscle were increased by 52% (p<0.05). GS activities in liver were decreased by up to 90% (p<0.01), a finding which undoubtedly reflects the loss of GS-rich perivenous hepatocytes following portal-systemic shunting. Immunoblotting techniques revealed no change in GS protein content of brain regions or muscle but a significant loss in liver of PCA rats. GS mRNA determined by semi-quantitative RT-PCR was also significantly decreased in the livers of PCA rats compared to sham-operated controls. These findings demonstrate that PCA results in a loss of GS gene expression in the liver and that brain does not show a compensatory induction of enzyme activity, rendering it particularly sensitive to increases in ammonia in chronic liver failure. The finding of a post-translational increase of GS in muscle following portacaval shunting suggests that, in chronic liver failure, muscle becomes the major organ responsible for the removal of excess blood-borne ammonia.     

First radiotherapy of human metastatic brain tumors delivered by a computerized tomography scanner (CTRx)

Purpose: This Phase I study was designed to evaluate the computed tomography (CT) scanner as a device for radiation therapy of human brain tumors (CTRx). This first use in humans of a modified CT for treatment was founded on extensive research experience with canine tumors. An additional objective was to increase the therapeutic radiation dose to tumors compared to normal tissue by concentration of infused contrast material in tumors, an effect available at diagnostic x-ray energies but not at megavoltage energies.

Methods and Materials: A small metastatic brain tumor in each of eight patients received 3–5-weekly fractions of 5 Gy equivalent per fraction from a CT scanner modified to deliver radiation therapy. In each patient, one additional tumor, lying completely outside the volume treated by CTRx, served as a control. The tumor receiving CTRx was treated after infusion of iodinated x-ray contrast media (CM) for dose enhancement. Many of these patients also received conventional 40 Gy whole brain radiation, before, during, or after CTRx treatment.

Results: None of the patients showed adverse reactions to the CM or necrosis of the normal brain from the CTRx boost radiation. Monte Carlo calculations of the radiation dose distributions in a model tumor showed that the CTRx irradiation of tumors carrying 10 mg or more of iodine per gram of tumor was as good or better than the dose distribution from conventional 10-MV X-rays. The treated tumor in two of the patients vanished after four treatments, whereas a control tumor in one patient remained constant and grew 4-fold in another patient.

Conclusion: The CTRx concept effectively combines a modified CT scanner as a diagnostic device, as a simulator dedicated to radiotherapy, and as a treatment machine. Thus, CTRx could be very useful for radiation oncologists in controlling CM-enhanced and other small brain tumors.     

Assessment of bone response to systemic therapy in an EORTC trial: preliminary experience with the use of collagen cross-link excretion. European Organization for Research and Treatment of Cancer.

This study was designed to evaluate new bone resorption and tumour markers as possible alternatives to serial plain radiographs for the assessment of response to treatment. Thirty-seven patients with newly diagnosed bone metastases from breast cancer, randomized to receive oral pamidronate or placebo tablets in addition to anticancer treatment within the context of a multicentre EORTC trial, who were both assessable for radiographic response in bone and had serum and urine samples collected for more than 1 month were studied. The markers of bone metabolism measured included urinary calcium (uCa), hydroxyproline (hyp), the N-telopeptide cross-links of type I collagen (NTx) and total alkaline phosphatase. The tumour markers measured were CA15-3 and cancer-associated serum antigen (CASA). Before treatment, levels of Ntx, uCa and Hyp were elevated in 41%, 24% and 28% respectively, and CA15-3 and CASA increased in 69% and 50%. For assessment of response and identification of progression, Ntx was the most useful bone marker. All markers behaved similarly in no change (NC) and partial response (PR) patients. There was a significant difference (P < or = 0.05) in Ntx levels (compared to baseline) at 1 and 4 months and in CA15-3/CASA at 4 months between patients with PR or NC and those with progressive disease (PD), and at 4 months between those with time to progression (TP) > 7 and those with TP < or = 7 months. The diagnostic efficiency (DE) for prediction of PD following a > 50% increase in Ntx or CA15-3 was 78% and 62% respectively. An algorithm to predict response to therapy has been developed for future prospective evaluation.     

Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform.

Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to the wt. Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not resistant to ICRF-187 compared to wt, whereas it was clearly less sensitive than wt to ICRF-187 at low ATP concentrations. This suggests that it is a shift in the equilibrium to an open-clamp state in the enzyme's catalytic cycle caused by a decreased ATP binding by the mutated enzyme that is responsible for bisdioxopiperazine resistance.     

Dietary glycine inhibits the growth of B16 melanoma tumors in mice.

Dietary glycine inhibited hepatocyte proliferation in response to the carcinogen WY-14,643. Since increased cell replication is associated with hepatic cancer caused by WY-14,643, glycine may have anti-cancer properties. Therefore, these experiments were designed to test the hypothesis that dietary glycine would inhibit the growth of tumors arising from B16 melanoma cells implanted subcutaneously in mice. C57BL/6 mice were fed diet supplemented with 5% glycine and 15% casein or control diet (20% casein) for 3 days prior to subcutaneous implantation of B16 tumor cells. Tumor volume was estimated from tumor diameter for 14 days. Tumors were excised, weighed and sectioned for histology post-mortem. B16 cells and endothelial cells were cultured in vitro to assess effects of glycine on cell growth. Statistical tests were two-sided and a P-value of 0.05 was defined as a significant difference between groups. Weight gain did not differ between mice fed control and glycine-containing diets. B16 tumors grew rapidly in mice fed control diet; however, in mice fed glycine diet, tumor size was 50-75% less. At the time of death, tumors from glycine-fed mice weighed nearly 65% less than tumors from mice fed control diet (P < 0.05). Glycine (0.01-10 mM) did not effect growth rates of B16 cells in vitro. Moreover, tumor volume and mitotic index of B16 tumors in vivo did not differ 2 days after implantation when tumors were small enough to be independent of vascularization. After 14 days, tumors from mice fed dietary glycine had 70% fewer arteries (P < 0.05). Furthermore, glycine (0.01-10 mM) inhibited the growth of endothelial cells in vitro in a dose-dependent manner (P < 0.05; IC50 = 0.05 mM). These data support the hypothesis that dietary glycine prevents tumor growth in vivo by inhibiting angiogenesis through mechanisms involving inhibition of endothelial cell proliferation.     

Kupffer cell oxidant production is central to the mechanism of peroxisome proliferators

Increased cell proliferation most likely plays a key role inperoxisome proliferator-induced liver cancer. Recently, Kupffercells were shown to be responsible for Wy-14,643-induced cellproliferation. However, the mechanism by which peroxisome proliferatorsactivate Kupffer cells is unknown. Since gut-derived endotoxinis a known activator of Kupffer cells, the hypothesis that itis involved was evaluated. Increased cell proliferation andperoxisome induction were unaffected by gut sterilization. Moreover,endotoxin was not detectable in portal blood following treatmentwith Wy-14,643. Therefore, it is concluded that gut-derivedendotoxin is not responsible for Kupffer cell activation. Totest the hypothesis that Kupffer cells are activated by Wy-14,643directly, Kupffer cell superoxide production was measured followingtreatment in vitro. Wy-14,643 increased superoxide productionin a dose-dependent manner (0.1 and 50 µM) with half-maximalstimulation at 2.5 µM. Diethylhexylphthalate (DEHP) andethylhexanol did not increase superoxide production even atdoses 50 times higher than Wy-14,643; however, monoethylhexylphthalate(MEHP) activated superoxide production as effectively as Wy-14,643with half-maximal stimulation at 5 µM. Treatment withWy-14,643 for 21 days caused a 2-fold increase in Kupffer cellsuperoxide production while DEHP did not. Pretreatment of Kupffercells with staurosporine (0.01–10 pM) completely blockedgeneration of superoxide demonstrating that protein kinase Cis required. Moreover, Wy-14,643 increased Kupffer cell proteinkinase C activity 3-fold. Pretreatment of Kupffer cells withthe amino acid glycine (0.01–3 mM), which blunts calciumsignaling, inhibited Wy-14,643-stimulated superoxide productionand increased protein kinase C activity completely. These dataare consistent with the hypothesis that potent peroxisome proliferators(Wy-14,643 and MEHP) directly activate Kupffer cell productionof oxidants via mechanisms involving protein kinase C. Further,peroxisome proliferator treatments that sustain elevated ratesof cell proliferation (e.g. Wy-14,643) activate Kupffer cellsuperoxide production following long-term dietary treatmentsupporting the hypothesis that Kupffer cell-derived oxidantsare involved in peroxisome proliferator-induced neoplasia.     

Fecal incontinence: a clinical approach

"Fecal incontinence" is defined as the involuntary loss of stool at any time of life after toilet training. It is a socially and psychologically devastating condition for patients and their families, and a topic which both patients and physicians are reluctant to approach. Although the true prevalence of fecal incontinence is unknown, studies have reported it to be as high as 2. 2% in the general population, with significantly higher rates among nursing home residents and hospitalized elderly. Risk factors include advancing age, female gender and multiparity. An understanding of pelvic floor anatomy and physiology is required to appreciate how diverse medical conditions can affect mechanisms involved in normal continence. The rectum serves as a storage reservoir until elimination can take place at a socially acceptable time and place. The pelvic floor muscles help to regulate the defecatory process and maintain continence. These muscles include the internal anal sphincter, the external anal sphincter and the puborectalis muscle. Each muscle contributes to normal continence, although the relative importance of each is controversial. Neurologic integrity and sensation are also key factors. Conditions associated with fecal incontinence include diarrheal states, fecal impaction, idiopathic neurologic injury, surgical and obstetric injury, pelvic trauma, collagen vascular disease, and neurologic impairment related to stroke, diabetes, or multiple sclerosis. Evaluation of the patient with fecal incontinence includes a directed history and physical examination, with particular attention paid to integrity of the perineum and rectum, and a complete neurologic evaluation. Diagnostic tools such as stool studies, anorectal manometry, defecography, electromyography, pudendal nerve conduction, and endoanal ultrasound may be employed in an outpatient setting. Fecal incontinence may be treated conservatively by employing such methods as dietary restriction, stool bulking agents, and biofeedback. Surgery may be the best option for cases refractory to medical treatment, or for those patients with rectocele or obstetrical injury. In this article, we review the presentation, epidemiology, pathophysiology, and etiology of fecal incontinence. Evaluation, including key components of directed history and physical examination, and the appropriate use of diagnostic studies and indications for treatment options are also addressed.