Standards in molecular diagnostics for the discovery and validation of clinically useful cancer biomarkers.

Recent discoveries in cancer biology have greatly increased the understanding of cancer at the molecular level, but translating this knowledge into clinically useful diagnostic tests has proved challenging. More efficient transfer of new molecular tests into patient care requires better standardization of laboratory practices, measurement methods and data management. The workshop assembled experts from National Cancer Institute, US FDA, National Institute of Standards and Technology, academia and industry, to address the most efficient approaches to biomarker standardization and validation.

The effect of prolonged hyperglycemia on metabolic alterations in the subtotally pancreatectomized rat.

A delayed onset of diabetes is characteristic of subtotally pancreatectomized patients in whom persistent hyperglycemia per se is documented to lead to the development of insulin resistance. This study was conducted to elucidate the metabolic alterations seen during transition of the acute to chronic phase after subtotal pancreatectomy (SP). Eight male Sprague-Dawley rats were studied 2 weeks after surgery in the acute phase, and the other eight at 4 weeks in the chronic phase.

Tumor-induced alterations in hepatic malic enzyme and carnitine palmitoyltransferase activity.

To better understand the role of the liver in the hypertriglyceridemia observed in a tumor-bearing state, we have examined tumor-induced alterations in hepatic lipogenesis and fatty acid oxidation. The effects of differing tumor burden as well as tumor excision on the activity and mRNA levels of malic enzyme and carnitine palmitoyltransferase were studied in Fisher 344 rats bearing a methylcholanthrene-induced sarcoma. Serum triacylglycerols and plasma nonesterified fatty acids (NEFA) levels were both elevated with increasing tumor burden (P < 0.

Organ blood flow in Fischer-344 rats bearing MCA-induced sarcoma.

Although blood flow is central to systemic metabolism, little is known about the effect of tumor on the perfusion of host tissues. This study evaluated the effects of a methylcholanthrene-induced sarcoma on blood flow to intra-abdominal organs and skeletal muscle of Fischer-344 rats anesthetized with pentobarbital sodium. Animals were studied by aortic injection of radiolabeled microspheres when the tumors reached 20% of body weight.

The effect of graded doses of insulin on peripheral glucose uptake and lactate release in cancer cachexia.

With the euglycemic clamp technique, we evaluated the effects of graded doses of insulin on glucose turnover rates and forearm lactate balance in five weight-losing patients with cancer before surgery and five age- and weight-matched healthy volunteers (control subjects). Insulin was infused sequentially at increasing rates of 0.5 (low physiologic), 1.

Effects of hypovolemia and transfusion on tumor growth in MCA-tumor-bearing rats.

Pretransplant blood transfusion has been shown to significantly affect the outcome of renal transplantation. Evidence regarding the association of blood transfusions with growth or recurrence of solid tumors is still conflicting both in clinical and in experimental studies, although diminished survival has been suggested in several studies. To determine the influence of blood transfusions and hypovolemia, as separate or combined factors, on tumor growth, we evaluated the weight of a subcutaneously implanted sarcoma (methylcholanthrene-induced) in 35 rats.

Tumor-induced alterations in tissue lipoprotein lipase activity and mRNA levels.

To elucidate the mechanisms of hypertriglyceridemia observed in the tumor-bearing rat, tissue lipoprotein lipase (LPL) activity and LPL mRNA levels were examined in the fed and fasted states at different degrees of tumor burden and after tumor removal. LPL activity in the epididymal fat pad and cardiac muscle in the 24-h-fasted rats was significantly decreased with increasing tumor burden (r = -0.53, P less than 0.

Metabolic alterations in obstructive jaundice: effect of duration of jaundice and bile-duct decompression.

We examined the effect of prolonged bile duct obstruction, and subsequent biliary decompression, on biochemical and metabolic parameters, using a reversible jaundice model in male Fischer 344 rats. The animals were studied after biliary obstruction for varying periods (4 days, one week, and two weeks) and following decompression. They were sacrificed one or two weeks following decompression.

Lipid kinetic alterations in tumor-bearing rats: reversal by tumor excision.

Cancer cachexia is frequently accompanied by hyperlipidemia. To identify the mechanisms underlying this alteration in lipid metabolism, free fatty acid (FFA) and very low density lipoprotein-associated triacylglycerol (VLDL-TG) kinetics were determined in tumor-bearing (subcutaneously implanted methylcholanthrene-induced sarcoma) Fischer 344 rats. The animals were studied after chronic vascular catheterization, in an unanesthetized, undisturbed state, after 24 hr of fasting.

A model of reversible obstructive jaundice in the rat.

A model of reversible, extrahepatic biliary obstruction is described. Vessel loop blockade of the biliary tree results in obstructive jaundice while removal of the exteriorized vessel loop provides internal biliary drainage without subsequent laparotomy. This technique combined with a system for chronic venous infusion and arterial blood sampling in the unrestrained rat is ideal for long-term metabolic studies of obstructive jaundice.

Intravenous glycerol infusions: effect on free fatty acid metabolism.

The plasma-free fatty acid response to intravenous glycerol infused at 250 and 500 mumol/min was determined in five normal volunteers in the postabsorptive state. There was a drop in free fatty acid concentration in all five subjects (one-way ANOVA, p less than 0.01) after the glycerol infusion with no change in insulin concentration compared to the post-absorptive state.

Expression of transcripts encoding a parathyroid hormone-related peptide in abnormal human parathyroid tissues.

A PTH-related peptide (PTHRP) has been identified and its cDNA cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The human PTHRP gene has been recently isolated and found to be a complex transcriptional unit using multiple promoters and containing alternatively spliced 3' exons which result in three mRNA classes, each class encoding a PTHRP with a unique carboxy-terminus. The PTHRP gene appears to be expressed in a number of normal tissues, and PTHRP transcripts have been previously reported to be overexpressed in a small sample of human parathyroid adenomas.

The reversal of increased gluconeogenesis in the tumor-bearing rat by tumor removal and food intake.

The accelerated mobilization of peripheral protein and subsequent increased gluconeogenesis are regarded as mechanisms of cancer cachexia. To determine the relation of gluconeogenesis to different degrees of tumor burden and subsequent tumor removal in the fed and fasted states, we examined the activity and mRNA levels of the key regulatory enzyme for gluconeogenesis: phosphoenolpyruvate carboxykinase (PEPck) in the liver of Fischer rats with a transplanted methylcholanthrene-induced sarcoma. PEPck activity in liver cytosol, after a 24-hour fast, was significantly higher in the tumor-bearing rats than in their pair-fed controls.

Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody.

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively.

Antibody against rat adipose tissue lipoprotein lipase.

To facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation, a high titre polyclonal antibody was raised against purified rat adipose tissue lipoprotein lipase (in a goat). The first stage of the purification of the lipoprotein lipase was carried out with heparin-Sepharose affinity chromatography. In the second stage we took advantage of the binding property of lipoprotein lipase to ampholytes.

Insulin regulation of fat cell ribosomes, protein synthesis, and lipoprotein lipase.

Ribosomes of high purity were isolated from fat cells by discontinuous sucrose gradient centrifugation. Approximately 23% of the ribosomes were membrane bound. A reproducible fraction of ribosomes was recovered as polysomes capable of incorporating [3H]leucine into peptides in a cell-free system.

Relation of body fat distribution to metabolic complications of obesity.

The importance of body fat distribution as a predictor of metabolic aberrations was evaluated in 9 nonobese and 25 obese, apparently healthy women. Plasma glucose and insulin levels during oral glucose loading were significantly higher in women with predominantly upper body segment obesity than in women with lower body segment obesity. Of the former group, 10 of 16 subjects had diabetic glucose tolerance results, while none of the latter group was diabetic.

Effects of insulin and procaine hydrochloride on glycogen synthetase activation and adipocyte calcium flux: evidence for a role of calcium in insulin activation of glycogen synthetase.

Observations that insulin-induced stimulation of glycogen synthetase occurs without detectable reduction in cyclic AMP suggests an alternative controlling mechanism. The hypothesis that this stimulation might be mediated through calcium was tested using procaine HC1, a drug whose insulin-like action is not associated with reduction in basal or adrenaline-stimulated cyclic AMP levels. Pretreatment of adipose tissue with insulin or porcaine for 30 minutes increased homogenate glucose-6-phosphate independent ("I") form activity to 58% and 48% respectively with no significant change in total activity.

Oestrogen induced hypertriglyceridaemia: role of the adrenal cortex.

The role of the adrenal cortex in the pathogenesis of hypertriglyceridaemia associated with the intake of oral contraceptive agents containing oestrogen has been investigated in rats. Bilateral adrenalectomy reduced the activity of hepatic enzymes regulating lipogenesis (acetyl CoA carboxylase, fatty acid synthetase) and decreased plasma triglyceride concentrations. On the other hand, the administration of high dosage corticosterone induced the activity of hepatic enzymes with consequent elevation in serum triglyceride levels.

The role of calcium in insulin action. IV. Mechanism of insulin resistance in adipose tissue of obese (ob/ob) mice and old Wistar rats.

The in-vitro antilipolytic response to insulin and procaine hydrochloride by adipose tissue from young rats (150 - 180 g) and lean mice has been compared with that from aged Wistar rats (600 g) and obese (ob/ob) hyperglycaemic mice. 1. The adipose tissue from the obese mice showed diminished responsiveness to insulin and to procaine hydrochloride.

The role of calcium in insulin action. III. Calcium distribution in fat cells; its kinetics and the effects of adrenaline, insulin and procaine-HCl.

The effects of adrenaline, insulin and procaine-HCl on Ca distribution in intact fat cells and on Ca binding to fat cell ghost membranes have been investigated. 1. Fat cells incubated in 45Ca containing media till isotopic equilibrium indicated that the exchangeable Ca in these cells averages 25.

Mode of insulin action.

A unifying hypothesis is proposed for the mechanism of insulin action in adipose tissue. Insulin both induces displacement of Ca++ from a membrane-bound pool and inhibits efflux of the ion, thereby facilitating a rise in intracellular free Ca++ concentration. The former effect could enhance the transport of substrates and ions into the cell, while the latter modulates the activity of some intracellular enzymes to stimulate glycogenesis, lipogenesis, and decrease lipolysis and glycogenolysis.