Molecular detection of neoplastic cells in lymph nodes of metastatic colorectal cancer patients predicts recurrence.

Disseminated disease, especially to the liver, constitutes the major risk of recurrence for colorectal cancer patients. However, successful resection can still be achieved in 25-35% of colorectal cancer patients with isolated metastases. To evaluate the clinical value of occult micrometastatic disease detection in lymph nodes, we tested genetic (K-ras and p53 gene mutations) and epigenetic (p16 promoter hypermethylation) molecular markers in the perihepatic lymph nodes from colorectal cancer patients with isolated liver metastases. DNA was extracted from 21 paraffin-embedded liver metastases and 80 lymph nodes from 21 colorectal cancer patients. K-ras and p53 gene mutations were identified in DNA from liver metastases by PCR amplification followed by cycle sequencing. A sensitive oligonucleotide-mediated mismatch ligation assay was used to search for the presence of K-ras and p53 mutations to detect occult disease in 68 lymph nodes from tumors positive for these gene mutations. Promoter hypermethylation at the p16 tumor suppressor gene was examined in both liver lesions and lymph nodes by methylation-specific PCR. Sixteen of the 21 (76%) liver metastases harbored either gene point mutations or p16 promoter hypermethylation. Twelve of the 68 lymph nodes were positive for tumor cells by molecular evaluation and negative for tumor cells by histopathology and cytokeratin immunohistochemistry, whereas none were positive for tumor cells by histopathology or negative for tumor cells by molecular analysis (P = 0.0005, McNemar's test). Moreover, in three patients with lymph nodes that were histologically negative at all sites, molecular screening detected tumor DNA at one or more lymph nodes. Survival analysis showed a median survival of 1056 days for patients without evidence of lymph node involvement by molecular analysis and 165 days for patients with positive lymph nodes by this approach (P = 0.0005). These results indicate that lymph node metastasis screening in colorectal cancer patients by molecular-based techniques increases the sensitivity of tumor cell detection and can be a good predictor of recurrence in colorectal cancer patients with resectable liver metastases.

De novo lipogenesis predicts short-term body-composition response by bioelectrical impedance analysis to oral nutritional supplements in HIV-associated wasting.

We studied the effects of enteral supplements on protein and energy intakes, body composition, energy expenditure, and gastrointestinal histology in 49 subjects with human immunodeficiency virus-associated weight loss (12.7 +/- 0.9% of body wt). We also determined whether a stable-isotope mass spectrometric measurement at baseline might predict the short-term response of fat-free mass (FFM) measured by bioelectrical impedance analysis. Thirty-nine subjects completed the study after being randomly assigned to receive either a whole-protein-based (n = 22) or a peptide-based (n = 17) formula. A nonsupplemented, nonrandomly assigned group (n = 13) was followed concurrently. Both formulas were well tolerated. Voluntary intakes of energy and protein from nonsupplement sources decreased significantly during supplementation [by 819-1638 kJ (196-382 kcal)/d and 5.6-14.4 g protein/d, respectively; P < 0.01] but to a lesser extent than the intake from the supplement [2300-2510 kJ(550-600 kcal)/d and 19-28 g protein/d, respectively], so that net increases in intakes of protein and energy (P < 0.03), as well as of several vitamins and trace elements were increased. Nevertheless, the mean FFM did not increase for the group as a whole, although there was considerable interindividual heterogeneity. Changes in FFM at 6 wk were significantly inversely correlated (r = 0.65, P < 0.01) with baseline synthesis of fat (de novo hepatic lipogenesis), but not with other potential measures of energy intake (insulin-like growth factor 1 or its binding protein) or inflammation (soluble tumor necrosis factor receptors I or II). The prospective identification of FFM response by measurement of de novo hepatic lipogenesis supported the hypothesis that the subset of wasting patients whose FFM is unresponsive to nutrient supplementation have altered nutrient metabolism.

Dying enterocytes downregulate signaling pathways converging on Ras: rescue by protease inhibition.

Organ and cell cultures of the small intestine serve as excellent in vitro models for programmed cell death (PCD). Cells cultured in serum-free, minimal medium rapidly died, as evidenced by histological changes, internucleosomal DNA cleavage, and TdT-mediated dUTP nick end labeling. Cell death was pervasive, although nonepithelial cells within the fibrovascular villus core were spared. PCD did not require a functional p53 gene. Serine and cysteine protease inhibitors, but not FCS, suppressed it. Relative to structural and functional proteins, dying enterocytes rapidly downregulated Ras-convergent proteins, including epidermal growth factor receptor, Erb-B2, and the son of sevenless guanine nucleotide exchangers. Reductions in the steady-state levels of both protein and mRNA were observed. These reductions were prevented by a combination of death-defying serine and caspase inhibitors, indicating a requirement for the initiation of death. Thus, during catastrophic PCD, intestinal epithelial cells delete cell surface signaling pathways responsible for Ras activation.

Weight loss, the gut and the inflammatory response in aids patients.

UNLABELLED: The objective of this study was to test the hypothesis that the integrity of the large bowel wall in AIDS patients is compromised in a manner that favours the chronic translocation of bacteria and/or products of bacterial metabolism into the bloodstream. When such translocation occurs, it induces a characteristic stress/inflammatory response in the body. Urinary butyrate, a unique product of colonic microbial metabolism, was used to assess gut wall permeability. Excretion of the pro-inflammatory cytokine IL-6 in the urine was used as a marker for the stress/inflammatory response. Four groups of subjects were studied, controls (n = 12), HIV + (n = 35) and AIDS patients with (n = 14) and without (n = 17) weight loss. RESULTS: measurable amounts of interleukin 6 (IL-6) and butyrate were found in the urine of all subjects. There were no significant differences in IL-6 excretion between the controls (0.68 +/- 0.64 pg/ml), asymptomatic HIV + subjects (0.59 +/- 0.37 pg/ml) and AIDS patients without weight loss (1.18 +/- 0.33 pg/ml) but IL-6 levels were significantly higher in the AIDS group with weight loss (4.02 +/- 1.26 pg/ml, P < 0.05). A similar pattern of results was found with interleukin 1 receptor antagonist (IL-1ra). Like IL-6 and (IL-1ra), urinary butyrate levels were increased in the AIDS patients with weight loss (2.83 +/- 0.67 mumol/l) relative to the controls (1.31 +/- 0.13 mumol/l, P < 0.05), with the HIV + patients (1.65 +/- 0.18 mumol/l) and AIDS patients without weight loss (1.90 +/- 0.22 mumol/l) falling in between. The data are consistent with a low, but chronic rate of bacteria and/or bacterial products seeping across a compromised colonic wall causing a chronic low stress response in AIDS patients.

The role of nutrition in AIDS management completes rite of passage.

AIDS: Presentations at the First International Conference on Nutrition and HIV Infection, held in April in Cannes, France, documented the positive role that objective multifactional nutritional support can have in managing disease complications. Overall, consensus was that the current system for accurate clinical evaluation of nutritional status, to the extent that it can serve as a basis for nutritional intervention that is relevant to patient outcome, lacks significant data and methods that are applicable to the HIV-positive population. However, nutritional intervention can have positive outcomes that are meaningful to the patient in terms of improved quality of life, and can be accomplished economically.^ieng

Long-term effects of early nutritional support with new enterotropic peptide-based formula vs. standard enteral formula in HIV-infected patients: randomized prospective trial.

Despite association with adverse clinical outcome, human immunodeficiency virus (HIV)-associated malnutrition has been relatively refractory to conventional nutrition management. Consequently, a prospective randomized trial was conducted to evaluate a new peptide-based enteral formula (NEF) in contrast to a standard enteral formula (SEF) in patients with HIV infection. Eighty early-stage largely asymptomatic patients were randomized into a dietary regimen supplemented with either a ready-to-feed NEF (18.7% protein, 65.5% carbohydrate, 15.8% fat; 1.28 kcal/ml) or SEF (14% protein, 55% carbohydrate, 31% fat; 1.06 kcal/ml). Patients received 2-3 8-oz cans of the NEF or SEF supplement per day for 6 mo. Parameters evaluated at 0 (baseline), 3, and 6 mo included adherence, weight change, anthropometric measurements, serum biochemical indices, gastrointestinal symptoms, physical performance, and intercurrent health events (including hospitalizations). For the 56 evaluable patients, those supplemented with NEF maintained their body weight significantly (p = 0.04) better, had significantly (p = 0.03) more stable triceps skin-fold measurements, and had significantly (p = 0.04) lower blood urea nitrogen than patients consuming the SEF supplement. Consumption of the NEF supplement was also associated with significantly reduced hospitalizations during the 3- to 6-mo evaluation period (p = 0.02). The NEF supplement was well tolerated and did not result in untoward clinical effects. These data suggest that supplemental use of an NEF provides superior nutritional management compared with an SEF for patients with early-stage HIV infection.

Chronobiology as it relates to toxicology, pharmacology, and chemotherapy.

Examples of circadian (daily) rhythms in man are presented and discussed. This is followed by presentation of experimental data indicating how the temporal organization affects the response of the host to potentially toxic agents, namely amphetamine and sodium pentobarbital. Data also are presented indicating that one unequivocally can improve therapeutic efficacy using the L1210 mouse leukemia model. This was accomplished by taking into consideration the circadian host toxicity response to anticancer agents, namely cytosine arabinoside, adriamycin, and cyclophosphamide. The subject monitored was the "cure rate" subsequent to treatment of different tumor loads.

Apoptosis in C3H/10T1/2 mouse embryonic cells: evidence for internucleosomal DNA modification in the absence of double-strand cleavage.

Apoptosis in embryonic C3H/10T1/2 (clone 8) cells is marked by specific changes in morphology and DNA fragmentation that differ from those found in apoptotic thymocytes. These results demonstrate that ultrastructural changes within the nucleus associated with endonucleolytic degradation are linked with structural degradation at higher levels of chromatin organization. Strand modifications within the internucleosomal linker region are shown to involve alkaline-sensitive sites that appear to be sensitive to S1 endonuclease. Our results suggest that apoptosis is not dependent upon internucleosomal cleavage and may reveal the penultimate step and the nature of the metabolic cascade that leads to cell death.

Historical comments on chronobiology with emphasis on cell proliferation, the concept of free running, cancer chemotherapy and experimental design.

A brief historical summary is presented regarding the emergence, over the past several decades, of chronobiology as the newest of the integrating discipline of biology. The emphasis is on the circadian system which normally is synchronized to the 24 h environmental light-dark cycle. In the absence of a suitable synchronizer, the system free runs on its own endogenous genetically determined frequency, which usually only approximates 24 h. Since the metabolic system changes rhythmically in time it follows that an organism such as man is biochemically and physiologically a different entity at different circadian stages; therefore it reacts differently to an identical stimulus given at different times. Different stimuli such as anticancer agents are examples considered clearly timed treatment has been shown to significantly improve therapeutic efficacy, data will be presented using the L1210 mouse leukemic model. Moreover data is presented showing that to ignore such rhythmic fluctuation when designing experiments that such can bring about experimental error and false interpretation. The common "same time of day" sampling does not take care of the rhythmic problem!

Diminishment of respiratory sinus arrhythmia foreshadows doxorubicin-induced cardiomyopathy.

BACKGROUND: The development of a microcomputer-based device permits quick, simple, and noninvasive quantification of the respiratory sinus arrhythmia (RSA) during quiet breathing. METHODS AND RESULTS: We prospectively and serially measured the radionuclide left ventricular ejection fraction and the RSA amplitude in 34 cancer patients receiving up to nine monthly bolus treatments with doxorubicin hydrochloride (60 mg/m2). Of the eight patients who ultimately developed symptomatic doxorubicin-induced congestive heart failure, seven (87.5%) demonstrated a significant decline in RSA amplitude; five of 26 subjects without clinical symptoms of cardiotoxicity (19.2%) showed a similar RSA amplitude decline. On average, significant RSA amplitude decline occurred 3 months before the last planned doxorubicin dose in patients destined to develop clinical congestive heart failure. CONCLUSION: Overall, RSA amplitude abnormality proved to be a more specific predictor of clinically significant congestive heart failure than did serial resting radionuclide ejection fractions.

Pharmacological and toxicological properties of arotinoids SMR-2 and SMR-6 in mice.

Studies were conducted to define primary pharmacological and toxicological properties of two arotinoids, SMR-2 and SMR-6, in male B6D2F1 mice. Mice were gavaged daily for up to 22 days with retinoids in corn oil (0.1, 0.2, or 0.4 mg/kg day SMR-2 or SMR-6 or 2.5, 10, or 30 mg/kg all-trans-retinoic acid as a reference control). Toxicological and biochemical endpoints were assayed after 8, 15, and 22 days. At toxic doses, i.e., those inducing weight loss, morphological changes were observed in skin, lymph nodes, spleen, bone marrow, liver, thymus, forestomach, adrenal, bone, and testes. Biochemical alterations included elevated serum alkaline phosphatase, corticosterone, and interleukins-1, -2, and -3. Additional immune alterations included increased responsiveness of spleen cells to both thymus-dependent and thymus-independent mitogens and increases in the total number of B cells in the spleen. At doses not inducing weight loss, target organ effects included the appearance of plasma cells and infiltration of polymorphonuclear cells in lymph nodes; myeloid cell hypercellularity in bone marrow; hematopoiesis in spleen; subacute inflammation in forestomach; and periportal cytoplasmic vacuolization in liver. At the low doses, SMR-2 resulted in decreased responsiveness of spleen cells to mitogens and SMR-6 caused increased responsiveness. SMR-6 also increased interleukin-1 and-2 production at low doses. Biochemical effects included reduced activities of liver aryl hydrocarbon hydroxylase (AHH) and soluble brain protein kinase C. Overall, the results suggest that leukopoiesis and reduced liver AHH and reduced soluble protein kinase C activities are the primary and initial pharmacological and toxicological effects of retinoids.

Retinoid receptor antisense DNAs inhibit alkaline phosphatase induction and clonogenicity in malignant keratinocytes.

Antisense oligodeoxynucleotides [oligo(dN)s] corresponding to human cellular retinol-binding protein I (cRBP) and human nuclear retinoic acid receptor alpha (hnRAR) were synthesized. Exposure of human malignant keratinocytes to these oligo(dN)s significantly attenuated the level of cytoplasmic cRBP and hnRAR in a concentration- and time-dependent manner. Further, the induction of alkaline phosphatase by retinol in these cells was blocked by treatment with 30 microM antisense oligo(dN) to cRBP or hnRAR but not by 30 microM of sense oligo(dN) to cRBP. Antisense oligo(dN) treatments concomitantly induced cell rounding, loss of cell-cell attachment, and cell adhesion to the substratum. By contrast, treatment of cells with an anticytokinetic agent, cytochalasin B, or with a cytostatic concentration of sodium azide failed to reduce cytoplasmic cRBP or hnRAR from nuclear extracts, even though antisense oligo(dN)-like changes in cell morphology were observed. Treatment of the cells for greater than 2.75 hr with 20-40 microM of either antisense oligo(dN) also led to the loss of clonogenic potential. These results show that both cytoplasmic and nuclear receptors for retinoids are important in the transduction of a retinoid signal response critical to cellular growth and differentiation. Our findings also suggest that defined genes, which are specified by retinoids and their receptors, may account for the pleiotropic effect of vitamin A compounds.

Tumorigenic and molecular characterization of novel phorbol ester-resistant and -sensitive lines of mice.

Two outbred lines of CD-1 mice were developed using males and females in an initiation (dimethylbenz[a]anthracene; DMBA), promotion (12-O-tetradecanoylphorbol-13-acetate; TPA) challenge, posttumorigenesis breeding protocol. Our results indicate that the phorbol ester-sensitive (PESTI) line developed tumors at a rate 4.1 times faster than the CD-1 parental line, while the phorbol ester-resistant (PERTI) line developed tumors at a rate 36 times slower than the CD-1 parents. The average number of tumors per mouse reached levels of 27.5 at 12 wk in the PESTI line, 0.1 at 16 wk in the PERTI line, and 6.7 at 16 wk in the CD-1 line. Biochemical tests showed that the PESTI line had both a high basal level and an enhanced epidermal ornithine decarboxylase (E.C. 4.1.1.17) response to TPA, the latter being nine times that of the PERTI line at their maximum dosages. An autoradiographic analysis of in vivo epidermal cell protein phosphorylation indicated marked differences in basal protein phosphorylation profiles (with high phosphate incorporation, PERTI, 112.7, 95.5, 64.4, 40.8, 18.6, 17.4, and 12.3 kDa; PESTI, 64.4, 40.8, 31.8, and 12.3 kDa) as well as TPA-dependent changes in these profiles (difference from basal levels, PERTI, 31.8 and 12.8 kDa; PESTI, 139.6, 126.3, 37.2, and 18.6 kDa). These heterogeneous profiles indicate strong genetic segregation of these protein kinase C target substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vitro characterization of the inhibition of mouse brain protein kinase-C by retinoids and their receptors.

The mechanism of the in vitro inhibition of Ca2+-, phosphatidylserine-dependent protein kinase C (PK-C)2 by the purified holo (ligand-saturated) forms of cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) was studied. We report here that the PK-C-inhibitory action of holo-cRBP and holo-cRABP is due to their respective ligands, all-trans-retinol and all-trans-retinoic acid; the reduced phosphorylation of the holo-retinoid-binding proteins and brain cytosolic proteins is not the result of a retinoid-induced soluble phosphatase or protease activity; retinoids reduce PK-C affinity for calcium and phosphatidylserine in vitro; and the structure-function activity of the retinoids and the specific interaction of these compounds with their binding proteins are important in blocking the activity of PK-C. These observations suggest that the inhibitory effect of retinoids on plasma membrane-associated PK-C activity pays a significant role in defining the early epigenetic aspects of PK-C-dependent tumor promotion and may be a physiological mechanism by which retinoids induce terminal differentiation in cell types that do not express soluble retinoid-binding proteins.

The in vivo inhibition of mouse brain protein kinase-C by retinoic acid.

The intracisternal injection of either all-trans-retinoic acid or [alpha]-difluoromethylornithine (DFMO) into the brain of 9-day-old mice blocked (greater than 90%) phorbol ester-induced ornithine decarboxylase (ODC, EC 4.1.1.17) activity in a concentration-dependent fashion; this inhibition was not evident with the use of the biologically impotent furyl analog of retinoic acid. In a similar manner, retinoic acid reduced the soluble protein kinase-C (PK-C) activity by 60% as well as total EGTA-sensitive kinase activity (66%) associated with the plasma membrane. Sixty-six percent of the retinoic acid-induced loss of PK-C activity in the soluble fraction could be accounted for by the translocation of PK-C to the plasma membrane as measured by the specific binding of 12-O-[3H]tetradecanylphorbol-13-acetate (TPA). DFMO and furyl-retinoic acid were not effective in altering PK-C activity or TPA binding to PK-C. In the presence of retinoic acid, however, there was a 2.3-fold increase in specific [3H]TPA binding in the plasma membrane fraction, which was 3.4-fold greater than that lost from the cytosol. Because retinoids do not directly affect TPA binding to PK-C, the data suggest that (i) the presence of retinoic acid results in the exposure of heretofore cryptic TPA-binding sites in the membrane, where this binding is most likely related to the alteration of membrane structure and (ii) de novo ODC induction is not required for retinoid-dependent inhibition of PK-C, although the TPA induction of PK-C appears to be necessary with regard to ODC induction.

Induction of mouse brain ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate is independent of TPA receptor concentration.

Ornithine decarboxylase (ODC, E.C. 4.1.1.17) activity was measured in a 35,000 X g brain supernatant fraction, prepared 5 h after intracisternal injection of 12-O-tetradecanoylphorbol-13-acetate (TPA) into developing mouse brain. TPA-dependent induction of ODC activity was maximal on days 5 and 9 postnatally while on day 7, the developmental (endogenous) level of ODC in brain was high and, concurrently, the ability of TPA to induce ODC was reduced. Both TPA-dependent and developmental increases in mouse brain ODC activity were significantly reduced by intracisternal injection of retinoic acid (RA). The efficacy of TPA in elevating ODC activity at postnatal ages 1-220 days-old was independent of both soluble-and particulate-associated TPA receptor concentration. These observations suggest that although TPA receptor activation may be an obligatory event in ODC induction, TPA receptor activation and its concentration per se, are not sufficient determinants for ODC induction and tumorigenesis. Furthermore, the endogenous mechanism of ODC induction is distinct from that of the TPA-dependent increase in ODC enzyme activity.

Retinoid-binding proteins are phosphorylated in vitro by soluble Ca+2- and phosphatidylserine-dependent protein kinase from mouse brain.

Cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) were purified from calf liver and uterus, respectively. Soluble Ca+2- phosphatidylserine-dependent protein kinase-C (PK-C) derived from mouse brain was capable of phosphorylating both of these apoproteins in vitro as determined by the phosphocellulose binding assay. The Km value was determined to be 6.2 microM for apo-cRBP and 5.1 microM for apo-cRABP. In contrast, the Km value for the histone III-S fraction was estimated to be 10.8 microM; the Km values for ATP in the presence of apo-cRBP and apo-cRABP were 12.4 microM and 2.6 microM, respectively. Specificity of phosphorylation of the retinoid-binding proteins was confirmed by polyacrylamide gel electrophoresis and subsequent autoradiography of the assay mixture as well as by a concentration-dependent, Ca+2, and phosphatidylserine sensitivity of the phosphorylation of both apo-cRBP and apo-cRABP. Inhibition of PK-C activity by holo-cRBP and holo-cRABP was also observed. Thus, phosphorylation of both of the retinoid-binding proteins may play an important modulating role in i) the ability of retinoids to function as antipromoters in chemically-induced tumorigenesis and ii) the control of physiological aspects of retinoid action in normal and retrodifferentiated cells.